In Vitro Anticariogenic Effects of Drymocallis rupestris Extracts and Their Quality Evaluation by HPLC-DAD-MS3 Analysis

In this study, for the first time, we investigated in vitro inhibitory effects of Drymocallis rupestris extracts and their subfractions obtained with solvents of different polarity (aqueous, 50% ethanolic, diethyl ether, ethyl acetate and n-butanolic) against bacterial viability and caries virulence factors of Streptococcus spp. strains. The diethyl ether subfraction (PRU2) showed bacteriostatic and bactericidal activity against mutans streptococci, with minimum inhibitory concentrations (MICs) in the range of 0.75–1.5 mg/mL and minimum bactericidal concentrations (MBCs) in the range of 1.5–3 mg/mL. Furthermore, PRU2 inhibited biofilm formation by Streptococci in a dose-dependent manner. It was also found that all five D. rupestris preparations exhibited diverse inhibitory effects on de novo synthesis of water-insoluble and water-soluble α-d-glucans by glucosyltransferases of the mutans group streptococci. The phytochemical profile of investigated samples was determined by spectrophotometric and chromatographic (HPLC-DAD-MS3) methods. The high polyphenol (total phenol, phenolic acids, tannins, proantocyanidins, and flavonoids) contents were found which correlated with anticariogenic activity of the analyzed samples. The results demonstrate that D. rupestris extracts and their subfractions could become useful supplements for pharmaceutical products as a new anticariogenic agent in a wide range of oral care products. Further studies are necessary to clarify which phytoconstituents of D. rupestris are responsible for anticaries properties.

phenolic content (Table 1), which varied from 29.9 ± 0.7 mg GAE/g dw for PRU2 to 30.9 ± 1.1 mg GAE/g dw for PRU3. It was obvious that the total phenolic content determined by Folin-Ciocalteu's method had not given a full characterization of the quality and quantity of the various groups of polyphenolics. The presence of different groups of phenolic compounds in investigated plant material was determined by weight and spectrophotometrical methods. The results ( Table 2) obtained showed that aerial parts of D. rupestris contained relatively high quantities of tannins (115.0 ± 3.7 mg/g dw) proanthocyanidins (4.6 ± 0.4 mg/g dw) and phenolic acids (7.8 ± 0.4 mg/g dw), as well as flavonoids (4.2 ± 0.3 mg/g dw) and (6.0 ± 0.5 mg/g dw), calculated as quercetin, flavonol type compound and parallel as a glycoside hyperoside, respectively.  It is noteworthy that according to our previous observations these differences in the values of total phenolic content (TPC) between all analyzed extracts and their subfractions can be attributed to the differences in their phytochemical composition. The total tannin content (TTC) determined was higher that this reported as the sum of all polyphenolic compounds (TPC). The significant difference found between this value arise from the use of two different analytical methods [15,16]. The observed significant differences between the total content of tannins (TTC) (155 mg/g) and condensed tannins (TPDC) (4.6 mg/g) in spectrophotometric assays was additionally confirmed by using the HPLC-DAD-MS 3 method. In fact, the hydrolysable tannins are predominant compounds both in the aerial parts as well as in the extracts from this plant. Forty five chemicals, numbered as 1-45, were detected ( Figure 1) and tentatively assigned as belonging to phenolic acid, tannin as well as flavonoid derivatives. The identification (Table 3) of all the compounds was carried based on comparison of their retention times, UV-Vis and MS spectra with chemical standards available or by comparison of UV-Vis, MS and MS/MS spectra with those found in literature. Numbering of the peaks is the same as in Table 3.

Anticariogenic activity
The effects of D. rupestris extracts and their subfractions on planktonic growth of Streptococci cells are shown in Table 4. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined based on micro-dilution method. Among the samples tested, the diethyl ether subfraction (PRU2) exhibited the significant bacteriostatic and bactericidal activity against all the tested bacteria with MICs of 0.75 or 1.5 mg/mL and MBCs of 1.5 or 3 mg/mL (see Table 4).
The effect of PRU2 on biofilm formation by the two selected strains, S. sobrinus 20381 and S. sobrinus/downei 21020, was examined by method similar for MIC assays for planktonic cells. The growth of S. sobrinus 20381 in a biofilm demonstrated higher susceptibility for PRU2 extract than the growth of S. sobrinus/downei 21020 ( Figure 2). However, the biofilm formation was almost completely inhibited at PRU2 concentration of 3 mg/mL. Thus, it was found that Streptococci cells in biofilm were somewhat more resistant to the diethyl ether subfraction compared to their planktonic form. It is probably related to higher biomass densities and decreased metabolic activities in biofilm, which affect the effectiveness of the therapeutic agent.    The structure of polysaccharides contained in the dental plaque matrix is the key factor in its ability to cause tooth decay, as it exerts an effect on the physical and biochemical properties of the biofilm. Matrix polysaccharides can increase microbial adhesion and biofilm cohesion, serve as an additional source of energy, protect microorganisms from hostile interactions, affect diffusion of substances to and from the biofilm, and facilitate concentration of ions of metals and other important nutrients in the biofilm environment. Among the great number of polymers synthesized by glucosyltransferases of cariogenic streptococci (1→6-α-D-, 1→4-α-D-and 1→3-α-D-glucans), the water-insoluble (1→3)-α-D-glucan plays a fundamental role in the etiology of dental caries because of unique properties that contribute to the formation of the plaque skeleton, i.e., it is easily adsorbed to saliva-or dental pellicle-coated enamel, promotes bacterial co-aggregation, and substantially enhances cohesion of plaque [17]. The effects of all tested preparations of D. rupestris on de novo synthesis of water-insoluble (IG) and water-soluble (SG) glucan by cell-free GTFs isolated from Streptococcus spp. were examined as a function of their concentrations and were expressed as the relative amount (%) of glucans produced at the certain extract or subfraction concentration compared to the amount produced in the absence of any plant substance ( Figure 3A and B).
It was found that all five formulations affected the synthesis of both types of glucans but to different degrees depending on preparation's concentration and streptococcal strain using as a GTFs source. In general, the degree of inhibition was proportional to the increasing concentrations of preparations, the effect of phytochemicals was more pronounced in the case of water-insoluble polymers. Glucan-synthesis activity of cell-free GTFs of S. sobrinus/downei 21020 and S. sobrinus 20381 was more strongly suppressed than the activity of GTFs of the other two strains. Both, PRU and PRU2 fractions showed the highest activity. A significant (60%) reduction of IG synthesis by S. sobrinus/downei 21020 was found at 0.75 mg/mL of PRU or PRU2. In the case of SG, the same effect was achieved only at a concentration of 6.0 mg/mL.

Preparation of Extracts and Their Subfractions
Different solvent systems (water, 50% ethanol, diethyl ether, ethyl acetate and n-butanol) were used to prepare the extracts and subfractions. Powdered plant material (2.0 g) was separately extracted with water (2 × 150 mL) -PRU or 50% ethanol (2 × 150 mL) -PRU1 in an ultrasonic bath (Sonic-5, POLSONIC, Warsaw, Poland) at controlled temperature (40 ± 2 °C) for 45 min. Supernatants were filtered through a funnel with glass wool, which was washed with 5 mL of solvent and concentrated to dryness under vacuum (Büchi Labortechnik System, Flawil, Switzerland) at controlled temperature (40 ± 2 °C), suspended in water and subjected to lyophilization using LABCONCO vacuum concentrator until a constant weight was obtained. Yields: PRU-79 mg; PRU1-92 mg.

Determination of Total Polyphenol Content
Total polyphenol content in extracts was determined at 765 nm (SPECORD 40, Analytik Jena, Jena, Germany) after the reaction with Folin-Ciocalteu's phenol reagent as gallic acid equivalents GAE/100g in mg per g of dry weight (dw) according to the manual colorimetric method described by Tawaha [18]. A sample aliquot of extract or subfraction (50 μL) was dissolved in distilled water (450 μL) and 0.2 N Folin-Ciocalteu's reagent (2.5 mL). After 5 min saturated sodium carbonate Na 2 CO 3 solution (75 g/L, 2 mL) was added. Samples were vortexed and incubated in the darkness at room temperature for 2 h. Quantitative measurements were performed, based on a standard calibration curve of different concentrations of gallic acid (20-500 mg/L). All measurements were performed in triplicate. The results are given in Table 2.

Determination of Total Phenolic Acids Content
Total phenolic acids content in plant material was determined by use the spectrophotometric method with Arnov's reagent according to the procedure described in Polish Pharmacopoeia IX [19]. Stock solution was prepared from the powdered sample (1.0 g) mixed with water (25 mL, two times) and shaken for 30 min each, then filtered. Phenolic acids were determined from a stock solution aliquot (1 mL) mixed with water (5 mL), hydrochloric acid (18 g/L, 1 mL), Arnov's reagent (1 mL) and sodium hydroxide solution (40 g/L, 1 mL) and diluted with water to 10 mL. Phenolic acids were measured spectrophotometrically at 490 nm. The percentage of phenolic acids, expressed as caffeic acid equivalents on dry weight, is calculated according to the formula: where A is the absorbance of the test solution at 490 nm and m mass of the powdered drug, in grams.
The results are given in Table 2 as means of experiments conducted in triplicate.

Determination of Total Flavonoid Content
The total content of flavonoids was determined by the spectrophotometric method by Christ and Müller [20] and followed the procedure described in Polish Pharmacopoeia IX [19]. Each powdered sample (0.6 g) was mixed with acetone (20 mL), 25% hydrochloric acid (281 g/L, 2 mL) and 0.5% urotropine (methenamine) solution (5 g/L, 1 mL) and heated in a water bath under reflux for 30 min. The obtained extract was filtered through cotton wool, and the sediment with the cotton wool was heated twice for 10 min with acetone (20 mL). The extracts were mixed and diluted with acetone to 100 mL in a volumetric flask. Then, this solution (20 mL) was diluted with water (20 mL), extracted with ethyl acetate (15 mL) and then, three time with ethyl acetate (10 mL). Organic phases were mixed and washed twice with water (40 mL), filtered into a volumetric flask and diluted with ethyl acetate to 50 mL. The obtained solution (10 mL) was added to four volumetric flasks. Then, to three flasks aluminum chloride (20 g/L -methanolic solution, 2 mL) was added and all four flask were filled with methanol-acetic aid glacial (19:1) to 25 mL. After hydrolysis, the flavonoids were measured spectrophotometrically at 425 nm by creating a complex with aluminum chloride in a methanol-ethyl acetate-acetic acid medium. The contents of total flavonoids, expressed as quercetin equivalent on dry weight, according to the formula , respectively: where A is the absorbance of the test solution at 425 nm and b the mass of the powdered drug, in grams. The results are given in Table 2.

Determination of Total Tannin Content
The total tannin content was determined by the weight method with hide powder according to the DAB 10 [21]. The results are given in Table 2.

Determination of Total Proanthocyanidin Content
The total proantocyanidin content was measured according to the European Pharmacopoeia [22]. Accurately weighted plant material (2.5 g) was heated under reflux for 30 min with ethanol (70% v/v, 30 mL). After that extract was filtered and the residue was flashed with ethanol (70% v/v, 10 mL). Then 25% hydrochloric acid (15 mL) was added with water (10 mL). The solution was heated under reflux for 80 min. After cooling, extract was filtered and filled up with ethanol (70% v/v) to 250 mL. Then the solution (50 mL) was evaporated to about 3 mL and transferred to a separatory funnel with water (15 mL). The solution was then extracted with n-butanol (3 x 15 mL, each). The organic layers were mixed and transferred to volumetric flasks and filled up with n-butanol to 100 mL. The absorbance was measured spectrophotometrically at 545 nm. The contents of proanthocyanidin, expressed as cyanidin chloride equivalent on dry weight, were calculated according to the formula: respectively, where A is the absorbance of the test solution at 545 nm and m the mass of the powdered drug, in grams. The results are given in Table 2.

HPLC-DAD-MS 3 Analysis
The HPLC-DAD-MS 3 analysis was performed using an UHPLC-3000 RS system (Dionex, Idstein, Germany) equipped with a dual low-pressure gradient pump, an autosampler, a column compartment, a diode array detector, and an AmaZon SL ion trap mass spectrometer with an ESI interface (Bruker Daltonik GmbH, Leipzig, Germany). HPLC analyses of extracts and fractions were carried out on a reversed-phase Zorbax SB-C18, 150 × 2.1 mm, 1.9 µm column (Agilent, Santa Clara, CA, USA). Column temperature was 25 °C. The mobile phase (A) was water/acetonitrile/formic acid (95:5:0.1, v/v/v) and the mobile phase (B) was acetonitrile/formic acid (100:0.1, v/v). A two-step gradient system was used: 0-60 min. 1%-26% B, 60-80 min 26%-50% B. The flow rate was 0.2 mL/min. The column was equilibrated for 10 min between injections. UV spectra were recorded over the range of 200-450 nm, chromatograms were acquired at 254 nm and 350 nm. The LC eluate was introduced directly into the ESI interface without splitting. Compounds were analyzed in negative and positive ion mode. The MS 2 fragmentation was obtained for two the most abundant ion at the time. The detection of neutral loses was set for the sugars moieties characteristic for glycosides fragmentation (132, 146, 162 and 176 amu). In the case of detection of one of the neutral loss masses the MS 3 fragmentation was performed in order to obtain the fragmentation spectrum of the aglycone moiety. The nebulizer pressure was 40 psi; dry gas flow 9 L/min; dry temperature 300 °C; and capillary voltage 4.5 kV. Analysis was carried out using scan from m/z 200 to 2.200.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
The D. rupestris preparations were tested for antibacterial activity by the broth dilution method. MIC values were determined in 96-well cell culture plates (Nunc, Roskilde, Denmark). A two-fold serial dilution of each sample in 20% DMSO was prepared. Plates with wells containing 180 μL of BHI medium plus 20 μL of 20% DMSO (100% growth controls) or 180 μL of BHI medium plus 20 μL of each dilution of test extracts (extracts final concentration from 12 to 0.09 mg/mL, DMSO final concentration-2%) were inoculated with a bacterial suspension containing 2 × 10 5 colony forming units/mL, and cultured for 24 h at 37 °C. MICs were determined as the lowest concentration of test samples that resulted in a complete inhibition of visible growth in the broth. After the determination of MICs, 10 μL aliquots of cultures were taken from wells showing no growth, inoculated into plates containing Mueller-Hinton Agar with 5% Sheep Blood (BBL), and cultured for 1 week. The minimum bactericidal concentrations were determined on the basis of the lowest concentration of the test extracts that kills 99.9% of the tested bacteria. Chlorhexidine digluconate (Sigma-Aldrich, St. Louis, MO, USA, final concentration from 25 to 0.08 µg/mL) was used as positive control to determine the sensitivity of each microbial species tested.

Inhibition of Biofilm Formation
Biofilms were cultivated on glass disks placed in a 24-well microtitre plates. Serial dilutions of each plant preparation in 20% DMSO were prepared and aliquots (50 μL) of each dilution were dispensed in wells of culture plates. Subsequently, 950 μL portions of BHI medium with 1% (w/v) sucrose were added, and 10 5 -10 6 of tested bacteria were inoculated into each well. Final concentrations of extracts ranged from 12 to 0.2 mg/mL, DMSO final concentration was 1%. The medium without extracts was used as a non-treated control. Chlorhexidine digluconate at 2.5 mg/L was used as a positive control. After incubation for 24 h at 37 °C, the disks were removed and media with unattached to substratum cells were removed by washing with phosphate buffered saline (PBS, pH 7.4). The minimum biofilm eliminating concentrations were determined as the lowest concentration of test samples that resulted in a complete inhibition of biofilm growth. For this purpose, three sets of disks were used to stain of the adhered biofilm with 1 mL of 1% erythrosine B for 5 min, rinsed thoroughly with water, and dried in room temperature. The bound dye was then removed from biofilms with 6 mL of 1 M NaOH. Biofilm formation was quantified by measuring optical density at 525 nm. The percentage of inhibition was calculated using the equation: (1 − A 525 of the test/ A 525 of non-treated control) × 100% (4)

Inhibition of streptococcal α-D-glucans synthesis
Crude preparations of Streptococcus spp. GTFs were prepared. Bacteria were grown in BHI at 37 °C for 18 h. Cell suspension was centrifuged (9600 × g, 30 min, 4 °C) and the culture supernatants were the source of crude extracellular GTFs. Supernatants were ultrafiltered and concentrated. To measure the inhibitory effects of extracts from D. rupestris on α-D-glucans synthesis we used a reaction mixture containing sucrose (30 mg), diluted GTFs (75 µL), and the dilution of each extract (75 µL, final concentration from 0.5 to 6 mg/mL) in 20% DMSO in a total volume of 1.5 mL of sodium phosphate buffer (pH 6.0) containing sodium azide to a final concentration of 0.05%. The medium without test substance was used as the non-treated control. After incubation for 24 h at 37 °C, the formed water-insoluble α-D-glucans were collected by centrifugation and washed twice with water. The water-soluble α-D-glucans were precipitated from the supernatant by addition of absolute ethanol (3 volume) followed by storage for 30 min at 4 °C and washed twice with 75% ethanol. Both glucans were determined utilizing phenol-sulphuric acid method with glucose as a standard [23].

Data Analysis
Statistical analysis was performed on all the three replicates from each treatment. Data were analyzed with Statistica 10.0. One-way analysis of variance was performed, followed by the Duncan test, for comparison of multiple means. The level of significance was p < 0.05.

Conclusions
The results of the study suggest that preparations from Drymocallis rupestris are promising natural products for the prevention of dental caries since they demonstrate antimicrobial activity against mutans streptococci and also inhibit in vitro the formation of dental plaque. These preparations contain huge quantities of polyphenols such as tannins, phenolic acids as well as flavonoids. Further studies are necessary to clarify which phytoconstituents from D. rupestris are responsible for the observed anticaries properties.