Chemical composition and antibacterial activity of Berberis vulgaris (barberry) against bacteria associated with caries

Abstract Objectives The aim of this in‐vitro study was to determine the antimicrobial capacity of a Berberis vulgaris plant extract on the bacteria being associated with caries including, Streptococcus mutans, S. sobrinus, S. sanguinis, S. salivaris and Lactobacillus rhamnosus. Material and methods Chlorhexidine 2% (CHX) mouthwash and ampicillin (10 μg/disk) were applied as positive control groups. Inhibition zone, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) related to stem, leaf and fruit of B. vulgaris plant were recorded for every five bacteria. Data were analyzed using SPSS ver. 22, one‐way ANOVA repeated measure and post hoc Tukey statistical test. The significance level was set at p < 0.05. Results There were no significant differences between the antimicrobial capacity of the positive controls and the extract from the stem and fruit of B. vulgaris (p > 0.05). The MIC values of the extract from the stem were significantly lower against S. sobrinus (64 μg/ml) and L. rhamnosus (128 μg/ml). The MIC value of the extract against S. mutans was significantly lower in the fruit group (64 μg/mL). The MBC value of the extract against S. sobrinus and L. rhamnosus was significantly lower in the stem group (128 μg/ml). The MBC value against S. mutans was significantly lower in the fruit group (128 μg/ml). Conclusions The results showed that CHX and B. vulgaris plant extract have similar antimicrobial activity against bacteria being associated with caries. Therefore, B. vulgaris, which shows antibacterial capacity, could be considered for further investigation as a safe, phytotherapeutic mouthwash to prevent dental caries.

Bacterial biofilm is the main etiological factor inducing dental caries (Allaker & Douglas, 2009). Destruction of dental structure occurred through the fermentation of sucrose and producing lactic acid by cariogenic bacteria (Yadav & Prakash, 2016). Streptococcus mutans (S. mutans) is the main bacterium of dental biofilm (Loesche, 1986). Lactic acid which produced by S. mutans, demineralizes hard dental tissues and this process makes the environment more suitable for other bacteria to grow (Westergren & Emilson, 1982).
Although the primary intervention to prevent dental caries is the mechanical removing of oral biofilm (Qiu et al., 2020) but application of antibiotics has also been suggested in a few studies to overcome dental caries (Lima et al., 2005). Antibiotics alone could not inhibit demineralization process occurred in dental biofilm and may lead to antibiotic resistance due to the formation of biofilm in dental biofilm (Kouidhi et al., 2015). Maintenance of ecological balance in microbial community and application of a combination of antimicrobial therapy with different mechanisms plays an important in control caries process (Yeke & Tao, 2012). Antibiotics change oral microbiota and contribute to development of resistant bacteria (Järvinen et al., 1993).
Other common side effects of antibiotics including vomiting, diarrhea and discoloration of teeth may also been observed (Chung et al., 2006).
Mouth washes are also recommended to control dental biofilm (Browning et al., 2000). Due to the frequent use of mouthwashes, the introduction of a material with minimal side effects (pigment formation on dental surfaces, infections, and tissue toxicity) and maximum beneficial effects (biofilm control) is essential (Amirzade-Iranaq & Masoumil, 2017;Wilson et al., 2014). In comparison to synthetic mouthwashes, herbal drugs have more economic efficiency besides fewer side effects (Bagheri et al., 2016).
Barberry (Berberis vulgaris L. a family of Berberidaceae) grows in Asia and Europe. The B. vulgaris is a shrub, about 1-3 m tall, with spiny, yellow wood and obviate leaves that have yellow flowers succeeded by oblong red berries (Ciulei et al., 1993;Damaschin, 2006;Dewick, 2002). Various parts of this plant, including the roots, bark, leaves, and fruit, have been used for medicinal purposes. The main alkaloids in this plant are berberine, berbamine and palmatine. Its main components have various therapeutic effects Imanshahidi & HJ, 2008;Javadzadeh & Fallah, 2012). Its powerful antimicrobial capacity against Staphylococcus aureus (S. aureus) and Candida spp. has been shown in the literature (Freile et al., 2003). Other in vitro studies have shown that the berberine is effective against Entamoeba histolytica, Giardia Lambia, Trichomonas vaginalis (Kaneda et al., 1991), Helicobacter pylori (Mahady et al., 2003) and Leishmania donovani (Ghosh et al., 1985).
In the range of studies conducted on B. vulgaris, the effect of this plant on dental caries microorganisms has not been studied and insufficient information is available in this regard. Therefore, the purpose of this study was to investigate the antibacterial effect of Barberry's extract on five common bacteria being associated with caries. Since the antimicrobial capacity of an extract depends on active ingredients, we have applied Gas chromatography-mass spectrometry (GC-MS) analysis to determine the chemical composition of the extract derived from the stem, leaf and fruit of Berberis plant.

| GC-MS analysis
For GC-MS analysis, the ground dried stem, fruit and leaf (2 g) of the plant were soaked in water (50 ml) at room temperature overnight.
Afterward, the aqueous solution was evaporated under reduced pressure and the dried extract was dissolved in ethanol and passed through a 0.45 μm filter before injecting into the chromatograph.
The Hewlett-Packard 5971 GC-MS device (Avondale, PA), available at the Islamic Azad University, Isfahan (Khorasgan) Branch, was applied to determine the chemical composition of the extract derived from the stem, leaf and fruit of Berberis plant. The GC-MS device had the following settings: 0.25 mm × 30 m polydimethylsiloxane DB-1 fused silica capillary column, 0.10-μm film thickness, 1 ml/minute carrier gas of helium, injector temperature of 250 C, and detector temperature of 200 C. The column temperature was set variable from 35 C/min to 180 C/min at 4 C V/min followed by 180 C/min to 280 C/min at 20 C V/min. The electronic impact of 70 eV was considered for the mass spectra.

| Screening of antimicrobial activity
The antimicrobial screening was performed using the disk diffusion assay (Andrews, 2001). A turbidity equivalent to 0.5 McFarland turbidity standard, holding 1.5 × 10 8 colony-forming units per milliliter (CFU/ml), was adjusted by inoculating colonies of fresh bacterial cultures into 5 ml of sterile broth medium, followed by inoculation of suspensions of Streptococcal strains and L. rhamnosus on Mueller-Hinton agar medium enriched with 5% defibrinated sheep blood (MHSBA; Merck, Darmstadt, Germany) and on De Man, Rogosa, and Sharpe (MRS) agar medium (Merck, Darmstadt, Germany), respectively. Sterile blank filters paper disks, 6 mm in diameter (Padtan Teb Co., Iran) were individually impregnated with 100 μl of the extract (10 mg/ml) to give a final concentration of 1 mg/disk and left to dry.
Extract disks were then placed on the seeded agar plates and were kept at 4 C for 1 h for diffusion of extract. Plates were incubated at 35 C for 24-48 h in 5% carbon dioxide (CO 2 ) condition. After incubation, the diameter (mm) of non-growth zones around disks was measured in comparison with 2% Chlorhexidine and ampicillin (10 μg/disk) as standards and normal saline as control. The test was repeated three times for each bacterial strain and extract. The inhibition zone was measured in millimeter, and the results recorded as mean ± SD. Data were analyzed statistically by ANOVA and Tukey HSD test.

| Determination of minimum inhibitory concentration and minimum bactericidal concentration
After confirmation of the antibacterial effect of B. vulgaris extract from stem, fruit and leaf, broth micro dilution method (Basri & SJIjoP, 2005) according to CLSI 2016 protocols was carried out to determine minimum inhibitory concentration (MIC) values. Plant extracts were suspended into DMSO 10% (which has no activity against test microorganisms) to make 256 μg/ml final concentration.
After that two-fold serially diluted and added to Mueller Hinton broth medium (MRS broth for L. rhamnosus strain) of 96-wells of microtiter plates. Hundred microliters bacterial inoculum (1 × 10 8 CFU/ml) was added to each well. Wells containing bacterial suspensions and broth media with extracts were used as negative and positive control, respectively.
The microtiter plates were incubated at 35 C for 24 h in 5% CO 2 condition. The well of the microtiter plate that showed no turbidity after incubation was taken as the lowest concentration of the extract (MIC value). The MIC value according to different extracts for each bacterial strain was assayed in triplicate.
The results of the antibacterial testing of B. vulgaris aqueous ethanolic extract from stem, fruit and leaf in comparison with Chlorhexidine and ampicillin are presented in Table 1 (the p-value recorded in Table 1 is only for the 1 mg/disk concentration of the extract).
Based on Tukey HSD test, in S. sobrinus bacterium, there was only significant difference between the antibacterial effect of leaf extract and CHX (p = 0.020). In S. sanguinis, S. salivaris and S. mutans there were only significant differences between the antibacterial effect of leaf extract and ampicillin respectively (p = 0.028, p = 0.015, p = 0.022). In L. rhamnosus, there was only significant difference between the antibacterial activity of fruit extract and ampicillin (p = 0.020).
Two-fold serial dilution ranges were between 8 and 256 μg/ml and the MIC value according to different extracts for each bacterial strain was assayed in triplicate. The MIC values listed in Table 2

| DISCUSSION
Dental caries is a multifactorial disease and bacteria play an important role in incidence and progression of this disease (Nishikawara et al., 2007). Clarke in 1927 isolated streptococci from human carious lesions and named them S. mutans and future studies revealed that Mutans streptococci (MS) and lactobacilli bacterium are involved in the development of dental caries (Clarke, 1924;Nishikawara et al., 2007).
Based on the results of the present study, the Berberis extract from the stem part of the plant showed the highest antimicrobial effect against S. sobrinus, S. sanguinis, S. salivaris and L. rhamnosus in comparison to fruit and leaf extract. In the present study, the Barberry stem extract showed antimicrobial efficacy against bacteria being associated with caries, comparable to CHX (a widely used mouthwash) and ampicillin as positive controls.
Berberine (the main alkaloid in the Berberis extract) showed a significant antibacterial and antifungal activity against Staphylococcus aureus (S. aureus) and different Candida spp. (Freile et al., 2003).
Antibacterial capacity of this extract against oral pathogens being associated with caries has not been studied up to the present (Imanshahidi & HJ, 2008). This extract has an inhibitory effect on the two virulence factors of sortase A (SrtA) and B (Srt B), that are mainly found in S. aureus (Oh et al., 2006). Sortase A enzyme that also produced with the S. mutans species is responsible for host cell attachment and biofilm formation (Wang et al., 2019). Berberine also interfere with the process of multi-drug resistant efflux pumps (mostly observed in S. aureus) and prevents the adherence of Group A streptococci to host cell (Stermitz et al., 2001).
In the present study, the MIC values of the stem extract against S. sobrinus and L. rhamnosus were lower in comparison to fruit and leaf extract. In contrast, the MIC value of the fruit extract was lower against S. mutans. With regard to MBC records, the stem extract T A B L E 1 In vitro comparison of antibacterial activity related to extracts from stem, fruit and leaf of B. vulgaris against cariogenic bacteria showed better results against S. sobrinus and L. rhamnosus, while against S. mutans, the MBC value was lower in the fruit extract group.
The extract concentration for antibacterial assay in the present study was determined as 10 mg/ml. Freile et al. (2003) in a study evaluated the antimicrobial activity of aqueous extracts of stem, root and leaf of Berberis and pure berberine against S. aureus, Enterococcus faecalis and different Candida spp. In the two extract concentrations of 500 and 1000 μg/ml that were applied in the Freile et al. (2003) study, no antibacterial activity was observed against the aforementioned bacteria. In contrast, pure berberine in concentrations of 50, 100 and 200 μg/ml showed high antibacterial activity against S.
aureus and Candida albicans. Since in the present study the extracts concentration was higher (10 vs. 500-1000 μg/ml), antibacterial activities were observed in all the experimental groups. It has been shown that the therapeutic dosage of B. vulgaris for most clinical situation is 200 mg orally, two or four times daily (Birdsall, 1997 (Balouiri et al., 2016). The filter blank papers utilized in this study had a good absorbability for the Berberis extract and could be applied to assess the inhibition of growth in the bacteria being associated with caries.
Environment is an important factor which can affect the phytochemical profile and antibacterial capacity of a plant at a given time.
Thus, geographic region of plant growth, the season of plant collection, climate, and plant storage conditions may influence the chemical composition and biological activity of a medicinal plant (Tabrizizadeh et al., 2018). Therefore, to avoid diverse values regarding a medicinal plant antibacterial effect, extract standardization is necessary.
The chemical and biological reactions of barberry's extract in the oral cavity require time to exert their effects on cell viability, and this will be the subject of a future study in order to standardize the preparation of barberry's extract as mouthwash. The intention is also to examine and compare the cytotoxicity and antimicrobial efficacy of the individual major metabolites present in the barberry's extract against important cariogenic bacteria in comparison to other interventions like fluoride therapy.
This novel approach investigated the antimicrobial activity of Berberis extracts in comparison to synthetic drugs. Nevertheless, for clinical application of this plant extract in preventive dentistry, more comprehensive studies are required to address the antibacterial activity in the clinical situation and effective concentrations of this medicinal plant against bacteria being associated with caries.

| CONCLUSION
The present study revealed that extracts obtained from stem and fruit of B. vulgaris show antimicrobial effects against cariogenic oral pathogens. These findings suggest that for clinical application of B. vulgaris as a safe, anticariogenic, and phytotherapeutic mouthwash, further investigations could be considered.

DATA AVAILABILITY STATEMENT
All data generated or analysed during this study are included in this article.