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The American Journal of Chinese Medicine, Vol. 42, No. 1, 157–171 © 2014 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X14500116

Withania somnifera Attenuates Acid Production, Acid Tolerance and Extra-Cellular Polysaccharide Formation of Streptococcus mutans Biofilms Santosh Pandit,* Kwang-Yeob Song† and Jae-Gyu Jeon* *Department †

of Preventive Dentistry Department of Prosthodontics School of Dentistry and Institute of Oral Bioscience Chonbuk National University Jeonju 561-756, Republic of Korea

Abstract: Withania somnifera (Ashwagandha) is a plant of the Solanaceae family. It has been widely used as a remedy for a variety of ailments in India and Nepal. The plant has also been used as a controlling agent for dental diseases. The aim of the present study was to evaluate the activity of the methanol extract of W. somnifera against the physiological ability of cariogenic biofilms and to identify the components of the extract. To determine the activity of the extract, assays for sucrose-dependent bacterial adherence, glycolytic acid production, acid tolerance, and extracellular polysaccharide formation were performed using Streptococcus mutans biofilms. The viability change of S. mutans biofilms cells was also determined. A phytochemical analysis of the extract was performed using TLC and LC/MS/MS. The extract showed inhibitory effects on sucrose-dependent bacterial adherence (100 g/ ml), glycolytic acid production ( 300 g/ml), acid tolerance ( 300 g/ml), and extracellular polysaccharide formation ( 300 g/ml) of S. mutans biofilms. However, the extract did not alter the viability of S. mutans biofilms cells in all concentrations tested. Based on the phytochemical analysis, the activity of the extract may be related to the presence of alkaloids, anthrones, coumarines, anthraquinones, terpenoids, flavonoids, and steroid lactones (withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxy withastramonolide). These data indicate that W. somnifera may be a potential agent for restraining the physiological ability of cariogenic biofilms. Keywords: Withania somnifera; Streptococcus mutans Biofilms; Dental Caries; Virulence Properties.

Correspondence to: Dr. Jae-Gyu Jeon, Department of Preventive Dentistry, School of Dentistry and Institute of Oral Bioscience, Chonbuk National University, Jeonju 561-756, Republic of Korea. Tel: (þ82) 63-270-4036, Fax: (þ82) 63-270-4035, E-mail: [email protected]

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Introduction Withania somnifera, known as Ashwagandha, is a plant of the Solanaceae family. It has been widely used as a remedy for a variety of ailments and a general tonic for overall health and longevity in Indian and Nepalese traditional medicine (Kataria et al., 2011; Bhattarai et al., 2013). The plant has also been employed traditionally for the treatment of toothache and tooth mobility (Mirjalili et al., 2009; Kharel et al., 2011). Recently, many pharmacological studies have revealed numerous biological activities of the plant, including antiinflammatory, anti-cancer, anti-stress, antioxidative, and immunomodulatory activities (Padmavathi et al., 2005; Mahdi et al., 2011). However, little is known about the biological effects of W. somnifera on dental diseases, especially on dental caries, even though it has been used as a controlling agent for dental diseases. Dental caries is a biofilm-related disease. If dental biofilms are allowed to remain on tooth surfaces with the frequent consumption of sugar, acidogenic bacteria will metabolize the sugar to organic acids (Marsh, 2003). The low pH environment in the biofilm matrix causes dissolution of the tooth surfaces and initiates dental caries. Although the oral microbial flora is quite diverse and complex, Streptococcus mutans in biofilms plays a major role in the development of the disease (Loesche, 1986); the bacterium can (1) adhere to and accumulate on the tooth surface, (2) utilize dietary sucrose to synthesize extracellular polysaccharides (EPS) of the biofilm matrix using glucosyltransferases (GTF), and (3) metabolize dietary sugars to organic acids and withstand rapid and substantial fluctuations in the environmental pH (Schilling and Bowen, 1992; Kuramitsu, 1993). The combination of these abilities allows S. mutans to effectively thrive in the complex oral microbiome and contribute to the development of dental caries. If the physiological ability of S. mutans in biofilms can be reduced, the acidification potential of dental biofilms and subsequent dental caries formation will be decreased. A widely adopted approach to prevent dental caries is the application of chemoprophylactic agents. These agents have attracted considerable attention and have played a crucial role in patient-directed approaches for dental biofilms control (Gaffar et al., 1997). However, some of these agents, e.g. chlorhexidine and antibiotics, have undesirable side effects including tooth staining and the emergence of bacterial resistance (Sreenivasan and Gaffar, 2002). Thus, increasing interest has been given to natural products as an alternative. Recently, we have reported that W. somnifera extract shows antimicrobial activity against S. mutans in the planktonic state (Pandit et al., 2013), suggesting that it could be a potentially useful anti-dental caries agent. However, little information is available on the activity of W. somnifera against the pathogenic bacterium in biofilms, even though bacterial cells growing in biofilms are generally believed to be physiologically and functionally distinct from planktonic cells (Hall-Stoodley et al., 2004). Considering the potential of W. somnifera as an anti-dental caries agent, it would be meaningful to evaluate the activity of the plant extract against the physiological ability of S. mutans in biofilms. The aim of this study was to evaluate the effect of methanol extract of W. somnifera on sucrose-dependent adherence, acid production, acid tolerance, and

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EPS formation of S. mutans in biofilms. In addition, this study investigated the chemical constituents of the extract. Materials and Methods

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Extraction and Phytochemical Characterization The powder of W. somnifera root was purchased from Dekha Herbals (Hattiban, Dhapakhel-1 Lalitpur, Nepal) and extracted using methanol, as previously described (Pandit et al., 2013). Preliminary phytochemical analysis was carried out according to the method of Wagner and Bladt (2001). The analysis was performed by thin layer chromatography (TLC; silica gel 60 F254 , Merck) using toluene:chloroform:methanol (3:2:1) as the mobile phase. Approximately 5l of MEWS was spotted onto the TLC plate. The resulting TLC spots were observed under UV254 nm and UV366 nm, before and after spraying with appropriate TLC reagents (Silva et al., 1998; Wagner and Bladt, 2001): Dragendorff reagent for alkaloids, 10% ethanolic KOH for anthrones and coumarines, Kedde reagent for cardenolides, Liebermann-Burchard reagent for triterpens or steroids, vanillin-sulphuric acid reagent for terpenoids, ninhydrin reagent for amino acids or amines, and AlCl3 for flavonoids. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was also performed to identify withanolide derivatives of W. somnifera. LC/MS/MS analysis was carried out using Xeno-TQS quantum access quadrupole mass spectrometer coupled to an ultra performance liquid chromatography (UPLC) and autosampler. Chromatographic separation was carried out by an ACQUITY UPLC® C18 column (1.7 m, 2:1  100 mm, Waters Technologies, Milford, MA, USA) at 30  C. The flow rate of the mobile phase was maintained at 0.5 ml/min and the injection volume was 5 l. The mobile phase A was 0.1% formic acid in deionized water, while the mobile phase B was 0.1% formic acid in acetonitrile. The gradient was as follows: 10% B at the initial stage for 1 min, increased to 100% B for 10 min and hold 100% B for 1 min, 10% B for 15 min and hold until 20 min. The source parameters were optimized by monitoring the MS and MS/MS spectra of the residues. The collision energy was optimized for each multiple reaction monitored transition. The instrument controls and data acquisition were carried out by the operating software of the masslynx V.4.1 data processing system (Waters Technologies, Milford, MA, USA). Each of the standards (withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxy-withastramonolide) and MEWS sample were dissolved in methanol and finally injected in a volume of 5 l. S. mutans Biofilm Formation S. mutans UA159 was used in the present study. S. mutans biofilms were formed on salivacoated hydroxyapatite (sHA) discs (2.93 cm2; Clarkson Chromatography Products, Inc., South Williamsport, PA, USA) and placed in a vertical position in 24-well plates, as

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detailed elsewhere (Pandit et al., 2012). Briefly, the sHA discs were generated by incubation with clarified human whole saliva for 1 h at 37  C. For biofilm formation, the sHA discs were transferred to a 24-well plate containing 1% sucrose (w/v) ultrafiltered tryptone yeast-extract (UTE) broth (pH 7.0) with S. mutans UA159 (2–5  10 6 colony forming units (CFU)/ml). The biofilms were grown in batch cultures at 37  C in the presence of 5% CO2 for 74 h. During the first 22 h, the biofilms were grown undisturbed to allow initial biofilm formation. From this point (22 h), the culture medium was changed twice daily (9 a.m. and 6 p.m.) until it was 74 h old.

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Biochemical Analysis for Acid Production, Acid Tolerance, GTF Activity and Viability Glycolytic pH-Drop Assay To evaluate the activity of MEWS against acid production and acid tolerance of S. mutans biofilm cells, a glycolytic pH-drop assay was performed as described elsewhere (Belli et al., 1995). Briefly, the 74 h old biofilms were transferred to a salt solution (50 mM KCl plus 1 mM MgCl2, pH 7.0) containing the vehicle control (4% DMSO) or MEWS (100, 300, 500 or 1000 g/ml). The pH was adjusted to 7.2 with 0.2 M KOH. Glucose (final concentration: 1% (w/v)) was then added. The decrease in pH was assessed over a period of 120 min using a glass electrode. The activity of MEWS against the acid production and acid tolerance was determined using the rate of acid production, calculated by the pH values in the linear portion (0–40 min), and the final pH values (120 min), respectively (Phan et al., 2004; Pandit et al., 2013). Acid Killing Assay The effect of MEWS on the acid killing of S. mutans biofilm cells was measured by the ability of the biofilm cells to survive at pH 2.5 (Xiao and Koo, 2010). For the acid killing assay, the 74 h old biofilms were dip-rinsed three times in 0.1 M glycine buffer (pH 7.0) to remove any residual culture medium. The biofilms were then incubated in 0.1 M glycine buffer (pH 2.5) containing the vehicle control (4% DMSO) or MEWS (100, 300, 500 or 1000 g/ml) for 30 and 60 min. After the respective incubation periods, the biofilms were dip-rinsed, removed and sonicated. The homogenized suspension was diluted serially and plated onto BHI agar plates to count the CFU. GTF Activity Assay The effect of MEWS on GTF activity was determined by measuring water-insoluble EPS (Song et al., 2006). The crude GTF of S. mutans were precipitated from the culture supernatant by adding solid ammonium sulphate and were then recovered, as detailed elsewhere (Song et al., 2006). The reaction mixture (total volume 1 ml) consisted of the following: (1) 300 l of the enzyme, (2) 175 l of 0.01 M potassium phosphate buffer (pH 6.8) containing NaN3 (3 mM), (3) 500 l of 0.01 M potassium phosphate buffer (pH 6.8) containing 5% sucrose and NaN3 (3 mM), and (4) the vehicle control (4% DMSO)

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or MEWS (100, 300, 500 or 1000 g/ml). NaN3 was added to prevent contamination by microorganisms. The reaction time was 24 h.

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Viability Determination To evaluate the activity of MEWS against the viability of S. mutans biofilm cells, the 74 h old biofilms were dip rinsed with autoclaved water and transferred into the media containing the vehicle control (4% DMSO) or MEWS (100, 300, 500 or 1000 g/ml). After 2 h incubation, the treated biofilms were removed and homogenized by sonication. The homogenized suspension was serially diluted and plated onto the BHI agar plate for determination of the CFU. Laser Scanning Confocal Fluorescence Microscopy (LSCM) Analysis for Bacterial Adherence and EPS Formation Assay for Sucrose-Dependent Bacterial Adherence The activity of MEWS against the sucrose-dependent adherence of S. mutans to sHA discs was examined by simultaneous in situ labeling of bacterial cells. For this assay, sHA discs were generated by incubation with clarified human whole saliva for 1 h at 37  C. The sHA discs were transferred into 1% sucrose UTE broth containing S. mutans UA159 (2–3  10 6 CFU/ml) with the vehicle control (4% DMSO) or MEWS (100, 300, 500 or 1000 g/ml) and incubated for 1 h. After incubation, the bacterial cells in sHA were labeled by means of 2.5 M of SYTO® 9 green-fluorescent nucleic acid stain (480/500 nm; Molecular Probes Inc., Eugene, OR, USA) for 20 min. LSCM imaging of the bacterial adherence was performed using the LSM 510 META (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium neon lasers. Two independent experiments were performed and 10 image stacks (512  512 pixel tagged image file format) per experiment were collected. The bacterial adherence was quantified from the confocal stacks by the image-processing software, COMSTAT (Heydorn et al., 2000). In this study, bio-volume and thickness of bacteria were analyzed to determine the differences among the treated biofilms. The bio-volume is defined as the volume of the biomass (m3) divided by the substratum (HA surface) area (m2). Assay for Bacterial Structure and EPS Formation The effect of a 2 h treatment of MEWS on the bacterial structure and EPS formation of S. mutans biofilms was examined using 74 h old biofilms. The 74 h old biofilms were transferred to new culture media containing 1 M of Alexa Fluor® 647-labeled dextran conjugate (10000 MW; absorbance/fluorescence emission maxima 647/668 nm; Molecular Probes Inc., Eugene, OR, USA) with the vehicle control (4% DMSO) or MEWS (100, 300, 500 or 1000 g/ml) and incubated for 2 h. The fluorescence-labeled dextran serves as a primer for GTF and can be incorporated into newly formed EPS by GTF during synthesis

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of the EPS matrix, but it did not stain the bacterial cells at concentrations used in the present study. After 2 h incubation, the bacterial cells in biofilms were labeled by means of 25 M of SYTO® 9 green-fluorescent nucleic acid stain. LSCM imaging of the biofilms was performed using the LSM 510 META. Two independent experiments were performed and 10 image stacks (512  512 pixel tagged image file format) per experiment were collected. The biofilms were quantified from the confocal stacks by COMSTAT. The biovolume and thickness of bacteria and the polysaccharides were analyzed to determine the differences among biofilms treated with the vehicle control or MEWS.

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Statistical Analysis All experiments, with the exception of LC/MS/MS and LSCM analysis, were carried out in duplicate, and each experiment was repeated at least four times. The data are presented as means  standard deviation. The intergroup differences were estimated by one-way analysis of variance, followed by a post hoc multiple comparison (Tukey test) to compare the multiple means. Values were considered statistically significant when p value was  0:05. The statistical analyses were performed using SPSS 12 software (SPSS Inc., Chicago, IL, USA). Results Chemical Characterization of the Extract Preliminary phytochemical screening by TLC indicated the presence of alkaloids, anthrones, coumarines, terpenoids, triterpenoids/steroids, and flavonoids in MEWS. In addition, withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxywithastramonolide were identified by LC/MS/MS analysis (Fig. 1). Effect on Sucrose-Dependent Adherence of S. mutans To investigate the effect of MEWS on biofilm formation, sucrose-dependent bacterial adherence assay was performed using LSCM. As shown in Fig. 2A, MEWS at all of the concentrations tested reduced the bio-volume of S. mutans on sHA discs by approximately 50%, compared to the vehicle control ( p < 0:05). However, the bacterial thickness was not affected by the treatment ( p > 0:05) (Fig. 2B). Figure 2C shows the representative LSCM images of S. mutans adherence to sHA discs at the concentrations of 0 (the vehicle control), 300, and 1000 g/ml. The number of S. mutans on sHA discs at 300 and 1000 g/ml was distinctly reduced compared to the vehicle control. Effect on Acid Production and Acid Tolerance of S. mutans Biofilms In the present study, the acid production and acid tolerance of S. mutans biofilm cells were affected by MEWS. As shown in the result of the glycolytic pH-drop assay (Fig. 3A), MEWS at  300 g/ml reduced the rate of acid production of the biofilms cells (p < 0:05).

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(A-1)

(A-2)

(B-1)

(B-2)

(C-1)

(C-2)

(D-1)

(D-2)

(E-1)

(E-2)

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Figure 1. Chromatogram of the standard of withanolide A (A-1), withanolide B (B-1), withaferin A (C-1), withanoside IV (D-1), and 12-deoxy-withastramonolide (E-1). A-2, B-2, C-2, D-2, and E-2 are the identification of the respective components in methanol extract of Withania somnifera using LC/MS/MS.

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A

B

C-1

C-3

C-2 Treated with VC

Treated with 300 µg/ml

Treated with 1000 µg/ml

Figure 2. Effect of methanol extract of Withania somnifera on the sucrose-dependent adherence of Streptococcus mutans UA159. Bacterial-bio volume (A) and thickness (B) of adhered bacteria to saliva-coated hydroxyapatite discs were quantified from the confocal stacks by the image-processing software COMSTAT. C-1, C-2, and C-3 are the representative images of adherence of bacteria in the presence of vehicle control (VC, 4% DMSO), 300, and 1000 g/ml, respectively. The data represent mean  standard deviation. Values followed by the same superscripts are not significantly different from each other ( p > 0:05).

The rate of acid production at the highest concentration tested (1000 g/ml) was approximately 56% lower than that of the vehicle control. Furthermore, the final pH values (at 120 min incubation) at the concentration of  300 g/ml were significantly higher than that at the vehicle control ( p < 0:05), reflecting that acid tolerance of S. mutans biofilm cells was disrupted by MEWS at  300 g/ml. To confirm the effect of MEWS on acid tolerance of the biofilm cells, an acid killing assay was performed. As shown in Fig. 3B, the number of S. mutans surviving cells in the biofilms treated with 1000 g/ml was significantly lower than that in the biofilm treated with the vehicle control at 30 and 60 min after acid exposure ( p < 0:05). However, the number of S. mutans surviving cells in the biofilms treated with  500 g/ml did not differ to that of the vehicle control ( p > 0:05). Effect on EPS Formation of S. mutans Biofilms As shown in Fig. 4A, MEWS did not directly affect GTF activity. However, the extract concentration-dependently reduced EPS formation of S. mutans biofilms (Figs. 4B, 4C and 4D).

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(B)

Figure 3. Effect of methanol extract of Withania somnifera on acid production and acid tolerance of Streptococcus mutans UA159 biofilm cells. (A) The change of the glycolytic pH drop pattern. The change of acid production rate and acid tolerance was calculated using the data of 0–40 min and final pH (120 min) respectively. (B) The change of S. mutans surviving cell CFU during acid challenge ( pH 2.5). The data represent means  standard deviation. The vehicle control (VC) was 4% DMSO. Values followed by the same superscripts are not significantly different from each other ( p > 0:05). A

B

C

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D-1 Treated with VC

900 µm

Treated with 300 µg/ml

900 µm

D-3

Treated with 1000 µg/ml

900 µm

Figure 4. Effect of methanol extract of Withania somnifera on glucosyltransferase (GTF) activity (A) and extracellular polysaccharide (EPS) formation of Streptococcus mutans UA159 biofilms (B, C, and D). The changes in EPS bio-volume (B) and thickness (C) were quantified from the confocal stacks by the imageprocessing software COMSTAT. The representative image of sample treated with the vehicle control (VC, D-1) showed a higher amount of EPS than those treated with 300 (D-2) and 1000 g/ml (D-3). The vehicle control was 4% DMSO. The data represent mean  standard deviation. Values followed by the same superscripts are not significantly different from each other ( p > 0:05).

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The bio-volume of newly formed EPS at  100 g/ml of MEWS was significantly less than that at the vehicle control ( p < 0:05) (Fig. 4B). Furthermore, the thickness of newly formed EPS at  300 g/ml of MEWS was lower than that of the vehicle control ( p < 0:05) (Fig. 4C). Figure 4D shows representative newly formed EPS images of S. mutans biofilms treated with 0 (the vehicle control), 300, and 1000 g/ml. In the images, the newly formed EPS bio-volume and thickness were concentration-dependently reduced by MEWS.

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Effect on Viability of S. mutans Biofilm Cells Figure 5A shows that the treatment of MEWS did not affect the viability of S. mutans biofilms cells. The number of CFU at the highest concentration of MEWS did not differ from that obtained at the vehicle control ( p > 0:05). Moreover, the treatment of MEWS did not affect the bacterial bio-volume and thickness of S. mutans biofilms (Figs. 5B and 5C). As shown in Fig. 5D, the representative image of biofilm treated with the vehicle control showed similar amount of bacterial cells, when compared to those treated with 300 and 1000 g/ml.

A

B

C

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D-1 Treated with VC

900 µm

Treated with 300 µg/ml

900 µm

D-3

Treated with 1000 µg/ml

900 µm

Figure 5. Effect of methanol extract of Withania somnifera on bacterial viability (A) and structure of Streptococcus mutans UA159 biofilms (B, C, and D). The changes in bacterial bio-volume (B) and thickness (C) were quantified from the confocal stacks by the image-processing software COMSTAT. The representative image of sample treated with the vehicle control (VC, D-1) showed similar amount of bacterial cells, when compared to those treated with 300 (D-2) and 1000 g/ml (D-3). The vehicle control was 4% DMSO. The data represent mean  standard deviation. Values followed by the same superscripts are not significantly different from each other ( p > 0:05).

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Discussion Natural products and their derivatives are still major sources of therapeutic agents for human diseases (Koehn and Carter, 2005). About 70% of all anti-infective agents introduced between 1981 and 2002 were derived from natural products (Newman et al., 2003). In the dental field, many researchers have also investigated the activity of natural products against oral diseases (Palombo, 2011). As part of an ongoing study on the prevention of dental caries by natural products, we focused on W. somnifera, which has been used traditionally for the treatment of dental diseases, and reported that the plant has bactericidal activity against S. mutans and S. sobrinus in the planktonic state (Pandit et al., 2013). However, little information is available on the activity of W. somnifera against the bacteria in biofilms, even though dental biofilms play an important role in the etiology of dental caries and bacterial cells growing in biofilms are generally believed to be distinct from planktonic cells (Stewart and Franklin, 2008). According to our knowledge, this study is the first to report the activity of W. somnifera against biofilms. Dental biofilms play an important role in the etiology of dental caries (Marsh, 2004). Thus, the control of dental biofilms is one of the most important targets for the prevention of the disease. To control the biofilms, several different approaches based on various underlying rationales have been used (Sbordone and Bortolaia, 2003): (1) inhibition of biofilm formation, (2) disruption of the physiological ability of existing biofilms, and (3) killing the microorganisms in biofilms. Although each of these approaches has been effective in its own right, many studies on the chemoprophylactic agents currently available for biofilm-related dental diseases have focused on the effects on the survival of bacteria in dental biofilms (Jeon et al., 2011). The focus on bactericidal effects appears to be logical considering that biofilms are principally composed of bacteria. However, the use of bactericidal agents can disrupt the resident microflora and the establishment of exogenous species, which can lead to pathological changes. Therefore, this study focused on the inhibitory effect of natural products on the physiological ability of biofilms cells. Since the minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) of MEWS against the bacterial strain used in the present study are 2 or > 4 mg/ml, based on our previous study (Pandit et al., 2013), we performed all of the assays at sub-MIC levels for the present study to eliminate the false positive results due to the bactericidal activity of the extract. In our previous study (Pandit et al., 2013), gas chromatography-mass spectrometry (GC/MS) analysis was performed to reveal the metabolite profiling of MEWS and the identified compounds were mainly primary metabolites, including mono and disaccharides, fatty acids and sugar alcohols. Since many secondary metabolites have been also identified and isolated from W. somnifera (Mirjalili et al., 2009; Kataria et al., 2011), we evaluated the presence of secondary metabolites in MEWS using TLC-reagents in this study. As expected, this study confirmed the presence of the alkaloids, anthrones, coumarines, terpenoids, triterpenoids/steroids, and flavonoids in MEWS. In addition, since many studies have reported that the major bioactive components of W. somnifera are steroidal alkaloids and lactones (a class of constituents together known as withanolides) (Kulkarni and Dhir, 2008), we also evaluated the presence of withanolides using LC/MS/MS. As shown in

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Fig. 1, our test extract also contained some withanolides (withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxy-withastramonolide). Based on the results of the chemical characterization, the activity of W. somnifera against the physiological ability of S. mutans biofilms could be related to the presence of alkaloids, anthrones, coumarines, terpenoids, triterpenoids, and flavonoids. Nevertheless, the precise bioactive constituents of W. somnifera are not revealed in this study. Thus, a more advanced bioactivity-guided study for isolation and purification will be needed to identify the precise active constituents against the biofilms. Many researchers have been interested in the inhibition of bacterial adherence to tooth surfaces since bacterial adherence is a prerequisite to dental biofilm formation and subsequent dental caries (Yu et al., 2007). As shown in Fig. 2, MEWS reduced the bio-volume of S. mutans on sHA discs after 1 h incubation despite no change in bio-thickness of the bacterium. This result clearly indicates that the sucrose-dependent adherence of S. mutans to sHA discs was affected by MEWS. Generally, it is well known that the sucrosedependent bacterial adherence in dental biofilms is closely related to GTF activity since the enzymes contribute to the formation of extracellular polysaccharides that allow the bacteria to attach to tooth surfaces (Loesche, 2007; Palombo, 2011). Interestingly, the inhibitory activity of MEWS against the sucrose-dependent adherence is not due to the reduction of GTF activity by MEWS. As shown in Fig. 4A, MEWS did not inhibit GTF activity. In the present study, the inhibitory effect of MEWS may result from a decrease in bacterial growth since the doubling time of S. mutans in the planktonic state was reduced by MEWS at the concentration of  1000 g/ml (Pandit et al., 2013). Acid production and acid tolerance are important virulence properties of S. mutans biofilms. In the present study, glycolytic pH-drop assay was performed to determine the effect of MEWS on the acid production and acid tolerance of S. mutans biofilm cells. In glycolytic pH-drop experiments, in which the cells are given excess glucose, S. mutans cells rapidly degrade glucose and lower the pH value of the suspension until they can no longer maintain a cytoplasmatic pH. Hence, the rate of the decrease in pH reflects the acid production capacity of S. mutans, whereas the final pH of the suspensions can reflect the acid tolerance of the bacterium (Gregoire et al., 2007). As shown in Fig. 3A, MEWS reduced the acid production of S. mutans biofilms cells. The inhibitory activity may result from the effect of MEWS on the bacterial glycolytic pathways rather than an inhibition due to a decrease in bacterial growth because resting bacteria were used in this assay. Figure 3A also shows the inhibitory effect of MEWS on the acid tolerance of S. mutans biofilm cells. Furthermore, the inhibitory effect of the test agents on the acid tolerance was confirmed by the result of an acid killing assay (Fig. 3B). However, the lowest effective concentration against acid tolerance in the acid killing assay (1000 g/ml) was higher than that in the glycolytic pH-drop assay (300 g/ml). This result may be due to the difference between the experimental methods. The effect of the test agent on acid tolerance in the acid killing assay was measured according to the number of surviving cells of S. mutans, but the effect of the test agent on acid tolerance in glycolytic pH-drop assay was measured by the physiological ability (acid production). Overall, it is apparent that MEWS could reduce the acid production and acid tolerance of S. mutans biofilms cells.

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It is well known that EPS are important components of the matrix in cariogenic biofilms and are recognized virulence properties involved in the pathogenesis of dental caries (Bowen and Koo, 2011). In the present study, MEWS reduced EPS formation of S. mutans biofilms (Figs. 4B, 4C and 4D), which suggests that the agent can modulate the development and accumulation of dental biofilms. According to previous studies, it was thought that the inhibitory effect of natural products such as green tea, propolis, cacao bean husk, and cranberry, on EPS formation was due to their activity against GTFs (Gregoire et al., 2007). However, MEWS did not reduce GTF activity in the present study (Fig. 4A). The possible mechanisms by which MEWS reduces EPS formation may involve its activity against the acid tolerance of S. mutans. Since enzyme secretion by bacterial cells is generally coupled to pH across the cell membrane (Marquis et al., 2003), it is possible that MEWS, which can affect pH by disrupting the acid tolerence of the biofilm cells, could affect the secretion of GTFs and thereby reduce the synthesis of EPS. Since our study focused on the inhibitory effect of natural products on the physiological ability of biofilms cells, we also investigated whether MEWS treatment for 2 h can affect the viability of S. mutans biofilm cells. Our findings suggest that MEWS in the concentrations tested did not exert bactericidal activity (Fig. 5A) or affect the bacterial bio-volume and thickness of S. mutans biofilms (Figs. 5B and 5C). This result suggests that MEWS treatment may not influence the growth of S. mutans biofilm cells since both the total number of CFU (live cells) and bacterial bio-volume and thickness of the biofilm cells (live plus dead cells) was not changed. Generally, our data clearly indicate that MEWS in the concentration tested could affect the physiological ability of S. mutans biofilm cells without exerting bactericidal activity, thereby reducing the virulence properties of S. mutans biofilm cells such as acid production, acid tolerance, and EPS formation. In conclusion, this study showed that MEWS is composed of alkaloids, anthrones, coumarines, terpenoids, triterpenoids/steroids, and flavonoids. Withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxy-withastramonolide were also identified in MEWS. MEWS showed an inhibitory effect on the virulence properties (sucrose-dependent bacterial adhesion, acid production, acid tolerance, and EPS formation) of S. mutans biofilms at sub-MIC levels. This result suggests that it might be useful for restraining the physiological activity of cariogenic biofilms and subsequently the development of dental caries. However, more advanced biochemical and phytochemical investigations are needed to identify its precise mechanisms and isolate the active components. Acknowledgments This work was supported under the framework of international cooperation program managed by the National Research Foundation of Korea (2012K2A1A2029691). References Belli, W.A., H.D. Buckley and R.E. Marquis. Weak acid effects and fluoride inhibition of glycolysis by Streptococcus mutans GS-5. Can. J. Microbiol. 41: 785–791, 1995.

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Withania somnifera attenuates acid production, acid tolerance and extra-cellular polysaccharide formation of Streptococcus mutans biofilms.

Withania somnifera (Ashwagandha) is a plant of the Solanaceae family. It has been widely used as a remedy for a variety of ailments in India and Nepal...
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