Ristiina Kaisu

Anu Riihinen

Marko Ryynanen

Eija Toivanen

Riitta Kononen

Carina Torronen

Tikkanen-Kaukanen

Kaisu Riihinen
email: kaisu.riihinen@uef.fi

Anu Ryynänen

Marko Toivanen

Eija Könönen

Riitta Törrönen

Carina Tikkanen-Kaukanen

S Mutans Ih

Naeslundii Ahp

Nucleatum Ahn

Antiaggregation potential of berry fractions

Keywords: berries, dental biofilms, antiadhesion, coaggregation, antiaggregation, 36 polyphenols

come

INTRODUCTION 38

Streptococcus, Actinomyces, and Fusobacterium are among the major bacterial genera, 39 which colonize the oral cavity early in life [START_REF] Könönen | Development of oral bacterial flora in young children[END_REF] and form crucial constituents of 40 dental plaque, i.e., dental biofilms accumulating on non-shedding tooth surfaces 41 [START_REF] Kolenbrander | 366 Bacterial interactions and successions during plaque development[END_REF]Rosan and Lamont, 2000). Dental biofilms play a crucial role in 42 the pathogenesis of the two most common oral infections, caries and periodontal diseases,

43
where intergeneric coaggregation contributes to the development of cariogenic and 44 periodontopathogenic communities [START_REF] Kolenbrander | 366 Bacterial interactions and successions during plaque development[END_REF]Sbordone et al., 2003). The 45 first step in the formation of dental plaque is the adhesion of early colonizers (streptococci 46 and Actinomyces) to the pellicle-coated tooth surface, while Fusobacterium nucleatum acts 47 as a bridge between early and late colonizers, coaggregating with most oral bacteria 48 examined (Al-Ahmad et al., 2007;[START_REF] Kolenbrander | 366 Bacterial interactions and successions during plaque development[END_REF]. Coaggregation is a further 49 adhesion process by which two genetically distinct bacteria become attached to one another 50 via specific molecules present in the biofilms. The basics of coaggregation have been 51 examined in vitro with the simple method of mixing bacterial suspensions together 52 [START_REF] Kolenbrander | 366 Bacterial interactions and successions during plaque development[END_REF].

53

While antimicrobial agents inhibit the survival and growth of bacteria, antiadhesion and 54 antiaggregation agents affect the formation of bacterial biofilms on oral surfaces. According 55 to [START_REF] Weiss | Inhibitory effect of a high-molecular-weight 419 constituent of cranberry on adhesion of oral bacteria[END_REF], the American cranberry (Vaccinium macrocarpon) constituents are 56 able to inhibit and reverse the coaggregation of dental plaque bacteria (antiaggregation). In 57 this context, information on other berries is still limited. Also, cranberry has been 58 demonstrated to inhibit oral bacterial adhesion in artificial biofilms (Steinberg et al., 2005; 59 [START_REF] Yamanaka | Inhibitory effects of cranberry juice on 422 attachment of oral streptococci and biofilm formation[END_REF][START_REF] Howell | Bioactive compounds in cranberries and their role in prevention of 359 urinary tract infections[END_REF]. Polyphenols are considered to be involved in nonspecific 60 antiadhesion effects against oral bacteria [START_REF] Duarte | Inhibitory 346 effects of cranberry polyphenols on formation and acidogenicity of Streptococcus 347 mutans biofilms[END_REF][START_REF] Ferrazzano | Anti-cariogenic effects 349 of polyphenols from plant stimulant beverages (cocoa, coffee, tea)[END_REF] Ltd (Kuopio, Finland). Bilberry, lingonberry, and crowberry (berry juice concentrates 83 (65°Brix) and cloudberries were purchased from Kiantama Ltd (Suomussalmi, Finland).

84

Cloudberry juice was prepared as described by Toivanen et al. (Toivanen et al., 2009).

85

Berry juices were stored at -20°C until used. The juices were fractionated into three 86 different fractions, <10 kDa fraction (FI), 10-100 kDa fraction (FII), and >100 kDa fraction 87 (FIII), through a molecular sieve by centrifugal filtering, as previously described (Toivanen 88 et al., 2009;2010). The soluble solids (SS) in the fractions were analyzed as ºBrix values 89 (SS g/100 g) using a digital refractometer (ATAGO PR-32, Tokyo, Japan) and diluted to the 90 fixed SS content of 12 g/100g, except for the cloudberry juice fractions, which were 91 analyzed as such.

92

Analyses of titratable acids and sugars. Titratable acids and sugars were analyzed based 93 on the methods developed for various foods [START_REF] Anon | Approved methods of the American Association of Cereal Chemists[END_REF]. Titratable acids were measured 94 from the juice fractions diluted in water by titrating with alkaline solution to their 95 potentiometric endpoint. The amounts of titratable acids were calculated by using the 96 equivalent weights of the major acids in berries (malic acid in apple, cloudberry, and 97 crowberry, and citric acid in bilberry, blackcurrant, and lingonberry). Sucrose was inverted 98 with acid hydrolysis prior to the analysis of reducing sugars with the Schoorl method 99 [START_REF] Anon | Approved methods of the American Association of Cereal Chemists[END_REF]. Reducing sugars were determined by using stabilized alkaline solution of a 100 copper salt (two Fehling's solutions). In the reaction, the amount of reduced copper is 105 Analysis of proanthocyanidins. Proanthocyanidin contents in the fractionated juices was 106 estimated by a spectrophotometric procedure [START_REF] Rohr | Analysis of procyanidins[END_REF], which was modified as 107 described by Toivanen et al. (2009). Briefly, juice fractions were diluted in acidified 108 methanol (0.6 M HCl) and the absorbance values of the samples were measured at 520 nm 109 with a UV-visible spectrophotometer (Shimadzu UV-1650PC; Kyoto, Japan) before and 110 after incubation at +70ºC (Memmert ULE 500 oven, Schwabach, Germany) for 3 h. The 111 absorbance differences of incubated and nonincubated samples were used for 112 semiquantification of proanthocyanidins as released anthocyanidins. The calibration curves 113 of proanthocyanidins were constructed by using depolymerized lingonberry dimer and 114 trimer isolate with purity of 80% [START_REF] Määttä-Riihinen | Catechins and 389 procyanidins in berries of Vaccinium species and their antioxidant activity[END_REF]. The range of the calibration 115 curve corresponded to 0.6-30% of proanthocyanidins of SS in the juice samples. (Määttä-Riihinen et al., 2004a;2004b;[START_REF] Määttä | Phenolic compounds in berries of black, red, 392 green, and white currants (Ribes sp.)[END_REF]. The 133 response factors for quantification of anthocyanins and flavonol glycosides were determined 134 from freshly prepared solutions of cyanidin-3-glucoside and quercetin-3-glucoside in the 135 concentration ranges 1.8-386 mg/l and 2-250 mg/l, respectively. Flavonol glycoside 136 concentrations lower than 2 mg/l were considered as non-significant (NS) (Table 1). The [START_REF] Kolenbrander | Coaggregations among oral bacteria[END_REF] 154 [START_REF] Mcintire | Inhibitors of coaggregation between 380 Actinomyces viscosus T14V and Streptococcus sanguis 34: beta-galactosides, 381 related sugars, and anionic amphipathic compounds[END_REF]. The coaggregating bacteria used in the assays were S. mutans with 155 either A. naeslundii (two strains) or F. nucleatum (two strains). In order to obtain four 156 parallel results, the antiaggregation assays were carried out in two runs. Berry juice

179

The coaggregation percent was calculated as follows:

180 181 ( ) ( ) ( ) 100 * % 2 1 2 1 2 1 A A A A A ion Coaggregat + - + = + , 182 183
where A 1 and A 2 present absorptions of the bacterial cell suspensions and A 1+2 presents 184 absorption of the coaggregation mixtures. The inhibition and the reversal activity induced 185 by the juice fractions (F) was expressed as percents and calculated as follows: (5.8% and 6.3 %, respectively), and in lingonberry FII and FIII (5.8% and 4.7%, 198 respectively). Although the anthocyanin contents were lower in fractions FI, the 199 chromatographic profiles of anthocyanins in the three fractions of the bilberry, blackcurrant, 200 crowberry, and lingonberry juices were identical in RP-HPLC-DAD analysis, as shown for 201 crowberry in Figure 1. Flavonol glycosides were distributed mainly in fractions FII and 202 FIII and major contents were 0.14% of SS in bilberry FIII, 0.09% of SS in blackcurrant FII, 203 0.05% of SS in blackcurrant FIII, and 0.07% of SS in crowberry FIII (Table 1). Titratable 204 acids and sugars were detected in comparable amounts in all the three fractions (Table 1). et al., 2005). The type of proanthocyanidin can influence the antiaggregation 278 ability (Foo et al., 2000). This concerns especially the high molecular size fraction FII of 279 lingonberry juice but also its weakly effective fraction FIII. In lingonberry and blackcurrant 280 juice fractions, the antiaggregation activity may result from the high content of 281 proanthocyanidins as well.

186 187 ( ) ( ) ( ) 100 * % 2 1 2 1 2 1 A A A A A ionF Coaggregat F F F + - + = + + +

282

It has been demonstrated that the polyphenol fraction (0.25 mg/ml of which 62% were 283 polyphenols) of American cranberry is able to inhibit the biofilm formation of two 284 periodontitis-associated anaerobes, Porphyromonas gingivalis and F. nucleatum (Yamanaka 285 et al., 2007). In another study, cranberry proanthocyanidins (0.500 mg/ml) and flavonols 286 (0.125 mg/ml), alone or in combination, reduced the formation and accumulation of S. 287 mutans biofilms [START_REF] Duarte | Inhibitory 346 effects of cranberry polyphenols on formation and acidogenicity of Streptococcus 347 mutans biofilms[END_REF]. In the present study, 0.6-1.4 mg/g of polyphenols in 288 bilberry, blackcurrant, crowberry, and lingonberry juice fractions displayed inhibition 289 and/or reversal activity against the studied coaggregates. The dilutions of the juice fractions 290 used in our antiaggregation assay represented the concentrations present in a drinkable 291 juice.

292

Berry juices can have a high content of fructose, glucose, citric acid, and malic acid 293 [START_REF] Viljakainen | Concentrations of organic acids and soluble sugars 413 in juices from Nordic berries[END_REF]. Therefore, a drinkable berry juice without further processing is 294 unsuitable for oral hygiene purposes. In the present study, considerable amounts of sugars [START_REF] Lussi | Inhibitory effects of cacao bean husk extract on plaque formation in vitro 378 and in vivo[END_REF]. Therefore, for clinical 299 studies or for possible clinical applications, antiaggregative berry polyphenols should be 300 isolated from the active fractions, as described for the high molecular weight non-dialyzable 301 material from cranberry juice concentrate [START_REF] Weiss | A high molecular mass cranberry constituent reduces 416 mutans streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite[END_REF]. In the case of polyphenolic 302 beverages, such as cocoa bean husk extract, red wine, cistus, and black tea, which have also 303 shown their potential in prevention of biofilm-induced diseases in the oral cavity, no further 304 processing is needed (Matsumoto et al., 2004;[START_REF] Hannig | Polyphenolic beverages 355 reduce initial bacterial adherence to enamel in situ[END_REF]. There is preliminary in 305 vivo evidence that daily usage of a mouthwash containing cranberry constituents or cacao 306 bean husk can reduce mutans streptococcal counts in the mouth (Matsumoto et al., 2004; 307 [START_REF] Weiss | A high molecular mass cranberry constituent reduces 416 mutans streptococci level in saliva and inhibits in vitro adhesion to hydroxyapatite[END_REF]. It has recently been reported that oral rinses with red wine, cistus tea, 308 and black tea can reduce the total count of adherent bacteria [START_REF] Hannig | Polyphenolic beverages 355 reduce initial bacterial adherence to enamel in situ[END_REF].

101

  proportional to the amount of reducing sugars in the sample. The determination was 102 continued by immediate titration with standard sodium thiosulphate solution until the end 103 was used as blank for the samples. The sugar content was read from a 104 conversion table based on the titer difference of the sample from the blank.

116

  Analyses of anthocyanins and flavonol glycosides. In order to analyze anthocyanins and 117 flavonol glycosides, the fractions were first diluted with methanol and acidified with 118 concentrated HCl to 0.6 M and then filtered through a 0.45-µm syringe filter (Pall Life 119 Sciences, Ann Arbor, MI) to the vials. High-performance liquid chromatography with diode 120 array detection (HPLC-DAD) apparatus consisted of a Hewlett-Packard (HP) instrument 121 with a 1100 series quaternary pump, an autosampler, and a diode array detector linked to an 122 HP-ChemStation data handling system (Waldbronn Analytical Division, Germany). HPLC 123 separation was achieved on a (150 x 4.6 mm i.d., 5 µm) Phenomenex Gemini reversed 124 phased (RP) column (RP-18, Merck, Darmstadt, Germany) protected with a guard column 125 of the same material (4 x 3 mm). The gradient program based on acetonitrile and methanol 126 (ACN:MeOH, 85:15, v/v) as the organic phase and 8.5% formic acid as the water phase was 127 modified from the procedure of Lätti et al. (2008). The gradual increase of the ACN-MeOH 128 of 1 ml/min was arranged as follows: 4-10% (0-2 min), 10% (2-20 min), 10-129 15% (20-35 min), 15-35% (35-40 min), 35-80% (40-50 min), 80% (50-52 min), return to 130 initial conditions in 8 min, and re-equilibration of the column for 10 min. Anthocyanins (at 131 520 nm) and flavonol glycosides (at 360 nm) were identified based on our earlier HPLC-132 DAD identification

  137 cyanidin-3-glucoside was measured in acidified mixture of methanol and water by using the 138 same solvent composition as used for the samples, since pH and water content affect the 139 absorption coefficient of anthocyanins. 140 Bacterial strains. Five clinical strains were selected, including Gram-positive S. mutans 141 IH 113728, A. naeslundii AHP 28639 and AHP 28651, and Gram-negative F. nucleatum 142 AHN 23952 and AHN 23937. The strains were grown in Brain Heart Infusion (BHI, LabM, 143 England) broth with 0.5% yeast extract (Difco) under anaerobic conditions at 37ºC. The 144 bacterial cells were harvested and washed with Dulbecco's phosphate-buffered saline (D-145 PBS) (10X stock solution, ECM4053, Eurolone) containing calcium and magnesium. The 146 washed cells were then either suspended in coaggregation buffer D-PBS to an optical 147 density of 1.5 at 400 nm (UV-Vis Spectrophotometer, GENESYS 10 Series, Thermo 148 Electron Corporation) for the coaggregation assay or stored at -20ºC in 50% glycerol 149 (99.5%, analytical grade, Normpur). The suspended cells were stored at 4ºC for the test 150 period of two weeks. 151 Coaggregation and antiaggregation assay. The inhibition and reversal activities of the 152 berry and fruit juice fractions against bacterial coaggregates were studied by using a turbidimetric assay developed by McIntire et al.

  157 fractions were diluted in D-PBS to the concentrations of 12 and 48 mg/g. Coaggregation 158 assays, inhibition and reversal of bacterial aggregates, and background measurements were 159 run simultaneously. In the inhibition assays, a 25 µl aliquot of each berry juice fraction was 160 mixed vigorously for 10 s with 25 µl of D-PBST (D-PBS with 0.05% (v/v) Tween20) and 161 with 25 µl of the cell suspension of S. mutans and A. naeslundii or F. nucleatum. In the 162 reversal test, 25 µl of the S. mutans cell suspension, 25 µl of the A. naeslundii or F. 163 nucleatum cell suspension, and 25 µl of D-PBST were vigorously mixed for 10 s. To allow 164 the formation of stable bacterial aggregates or bacterium-berry coupling, the mixtures were 165 then agitated gently (100 revolutions per minute) in an orbital shaker (Orbital Shaker, Stuart 166 Scientific, United Kingdom) for 30 min. Then the other bacterial pair (25 µl of the cell 167 suspension) was added to the inhibition test mixtures and berry juice fraction (25 µl) was 168 added to the reversal test mixtures. For background measurements, two different blanks 169 were made. The blanks were prepared by mixing 25 µl of each bacterial cell suspension 170 with 50 µl of D-PBST and 25 µl of the berry fraction as well as by mixing 25 µl of each cell 171 suspension with 75 µl of D-PBST. The coaggregation score for each bacterial pair was 172 measured from the mixture containing 25 µl of the both cell suspensions and 50 µl of D-173 PBST. All the prepared samples and blanks were mixed vigorously, agitated gently for 30 174 min, as described above, and then 200 µl of D-PBST was added. The mixtures were then 175 centrifuged at 200 rpm (Eppendorf centrifuge 5415D, Hamburg, Germany) for 10 min to 176 pack down the aggregates. The top part of the supernatant was transferred to a flat-bottomed 177 The turbidity was measured at 630 nm by using a microtiter plate reader 178 (Victor2, 1420 Multilabel counter, Wallac, PerkinElmer LifeSciences, Boston, USA).

205

  Coaggregation and antiaggregation assays. The degree of coaggregation was relatively 206 low, being equal between S. mutans IH 113728 and the two strains of A. naeslundii (AHP 207 28639 and AHP 28651) and between S. mutans IH 113728 and the two strains of F. 208 nucleatum (AHN 23952 and AHN 23937) (Table2). Inhibition and reversal activities less 209 than 50% were ignored, as proposed by[START_REF] Mcintire | Inhibitors of coaggregation between 380 Actinomyces viscosus T14V and Streptococcus sanguis 34: beta-galactosides, 381 related sugars, and anionic amphipathic compounds[END_REF]. All positive scores for 210 inhibition and reversal (antiaggregation) activities displayed low standard deviations and 211 were reproducible in the four experiments. The reversal and inhibitory activity was found in 212 FII and FIII of bilberry, blackcurrant, crowberry, and lingonberry juices (Table 2). The 213 antiaggregation activity was detected in all eight bacterial pairs with the fraction FII of 214 , and lingonberry juices. In addition, the crowberry juice fraction FIII 215 had antiaggregation activity to seven of the eight studied pairs. Of the blackcurrant juice 216 fractions, FIII was the most active but the effect was limited to the four bacterial pairs of S. 217 mutans and A. naeslundii. The blackcurrant FII fraction had effect only to one of the studied 218 eight bacterial pairs. The fractions FIII of bilberry juice and lingonberry juice showed 219 activity to three and one of the eight bacterial pairs, respectively. The antiaggregation was 220 mainly achieved with the berry concentration of 48 mg/g of SS. The crowberry juice 221 fraction FIII with the concentration of 12 mg/g of SS gave reversal activity to two of the 222 bacterial pairs and that of blackcurrant induced aggregation inhibition of one bacterial pair. 223 The inhibitory and reversal activity of 100% was detected in the FII fraction of bilberry 224 juice with the concentration of 48 mg/g of SS. The fractions of apple and cloudberry juices 225 exhibited no antiaggregation activity. , it is not due to the specific anthocyanin structures in the 274 studied bilberry, blackcurrant or crowberry juice fractions. 275 Among berries, there are two different types of proanthocyanidins: A-type (rare) and B-276 type (common). The rare A-types dominate in the Finnish Vaccinium berries (Määttä-277 Riihinen

  295and titratable acids were present in all the three fractions examined. Low molecular weight 296 by acid-producing dental bacteria in plaque, which may increase the 297 risk of dental caries. Moreover, the low pH value and titratable acidity are the major 298 chemical risk factors for dental erosion

309Figure 1 .

 1 FIGURE LEGEND

435aFractions

  FIGURE 1

  ;

	82	solids g/100 g of solution) and blackcurrant (65ºBrix) were obtained from VIP-Juicemaker
		F o r F o r
		P P
		e e
		e r e r
		R R
		e e
		v i e v i e
		w w
	61

10 RESULTS 190 Composition of the juice fractions.

  + The contents of proanthocyanidins and anthocyanins 191 in the juice fractions varied between different berry species (Table1). The low molecular

	192
	193	size fractions (<10 kDa in FI) and all the three fractions of apple and cloudberry displayed
	194	low amounts of proanthocyanidins and no anthocyanins. The anthocyanin concentration
	195	was high in bilberry FII and FIII (3.99 % and 5.21 % of SS, respectively) and in crowberry
	196	FII and FIII (2.80% and 5.18 %, respectively). The proanthocyanidin concentration was
	197	r o F high in blackcurrant FII and FIII (8.5% and 6.2%, respectively), in crowberry FII and FIII
							P
							e
	188						e r
	189	Inhibition	/	reversal	%	=	% Coaggregat ion Coaggregat % ion Coaggregat -R	ionF	%	*	100
							e
							v i e
								w

TABLE 1 .

 1 Composition of soluble solids (SS) a in indicated juice fractions.

	434
				% of SS
		SS g/100g TA	Sugar	PA	AC	FG
	Apple
	FI	22	4.5	70	<0.6	ND c	ND
	FII	24	4.6	77	<0.6	ND	ND
	FIII	24	4.5	77	<0.6	ND	ND
	Bilberry
	FI	18	11.2	80	<0.6	0.34	ND
	FII	24	11.9	63	1.9	3.99	NS
	FIII	32	12.7	83	1.4	5.21	0.14
	Blackcurrant
	FI	17	24.4	63	0.7	0.29	ND
	FII	25	22.3	59	8.5	2.01	0.09
	FIII	24	22.2	58	6.2	1.31	0.05
	Cloudberry
	FI	3	18.9	57	<0.6	ND	ND
	FII	4	19.1	65	<0.6	ND	ND
	FIII	3	NA	NA	<0.6	ND	ND
	Crowberry
	FI	16	7.4	65	0.7	0.29	ND
	FII	22	7.6	55	5.8	2.80	NS
	FIII	32	7.5	48	6.3	5.18	0.07
	Lingonberry
	FI	19	17.4	52	1.2	0.09	NS
	FII	25	16.4	53	5.8	0.32	NS

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The in vivo dynamics of Streptococcus spp., Actinomyces naeslundii,

ACKNOWLEDGEMENTS

322

Kiantama Ltd. (Suomussalmi, Finland) and VIP-Juicemaker Ltd. (Kuopio, Finland)


Michal Tomczyk
email: tomczyk@umwb.edu.pl

Adrian Wiater

Malgorzata Pleszczyńska

Michał ; Tomczyk

Michał Tomczyk

Małgorzata Pleszczyńska

Michał Tomczyk

In vitro Anticariogenic Effects of Aerial Parts of Potentilla recta and Its Phytochemical Profile

Keywords: Potentilla recta, extracts, polyphenolic compounds, anticariogenic activity Potentilla recta, extracts, polyphenolic compounds, anticariogenic activity Anticariogenic Effects and Phytochemical

In this study, for the first time, we investigated in vitro inhibitory effects of Potentilla recta extracts and subfractions obtained with solvents of different polarity (aqueous, 50% ethanolic, diethyl ether, ethyl acetate and n-butanolic) against cariogenic Streptococcus spp. strains. It was found that the tested samples inhibited the growth of oral streptococci. Furthermore, all five P. recta preparations exhibited an inhibitory effects on water-insoluble α-(1→3)-, α-(1→6)-linked glucan (mutan) and artificial dental plaque formation. The ethyl acetate fraction showed the highest anti-biofilm activites especially against S. sobrinus GCM 20381, with a minimum mutan and biofilm inhibition concentrations of 6.25 and 25 µg/mL, respectively. The phytochemical profile of active constituents in the investigated samples were analyzed. The high polyphenolics (total phenol, phenolic acids, tannins, proantocyanidins, flavonoids) content were found. The ethyl acetate fraction showed the

INTRODUCTION

Dental caries is an infectious disease which is widely distributed throughout the world and is the most prevalent chronic oral disease in humans. The formation of dental caries is caused by the colonization and accumulation of oral pathogens and extracellular polysaccharides (dextrans and mutans) that are synthesized from sucrose by glucosyltransferases of mutans streptococci [START_REF] Pleszczyńska | Searching for natural substances inhibiting glucosyltransferases from mutans streptococci[END_REF]. Numerous synthetic substances and antibiotics have been used in the control of dental plaque. However, these compounds cause many unexpected side-effects [START_REF] Sato | Antibacterial property of isoflavonoids isolated from Erythrina variegata against cariogenic oral bacteria[END_REF][START_REF] Park | Kuwanon G: an antibacterial agent from the root bark of Morus alba against oral pathogens[END_REF]. Recently, the use of medicinal plant and natural products, incorporated in food and beverages, has been claimed to offer potential candidates for anticariogenic agents. Among these phytoconstituents, several polyphenolic compounds like tannins, flavonoids and also alkaloids seem to be the most promising biomolecules [START_REF] Eley | Antibacterial agents in the control of supragingival plaque -a review[END_REF][START_REF] Smullen | The antibacterial activity of plant extracts containing polyphenols against Streptococcus mutans[END_REF][START_REF] Petti | Polyphenols, oral health and disease: A review[END_REF].

The genus Potentilla L. (Rosaceae) has been known since ancient times for its curative properties. Extracts of the aerial and/or underground parts have been applied in traditional medicine for the treatment of inflammations, wounds, certain forms of cancer, infections, diarrhoea, diabetes 3 mellitus and other ailments. Most of the pharmacological effects can be explained by the high amount of polyphenolic compounds (hydrolyzable and condensed tannins, flavonoids) and also triterpenoids [START_REF] Tomczyk | Potentilla -A review of its phytochemical and pharmacological profile[END_REF][START_REF] Tomczyk | Potentilla -A review of its phytochemical and pharmacological profile[END_REF].

Sulphur cinquefoil (Potentilla recta L.) is a long-lived invasive perennial plant from Eurasia that has become one of the most serious invaders of natural area grasslands in North America and Canada. P. recta is a perennial herb which widely inhabits well-drained soil in full sun to part shade, and prefers calcareous soils. It is found in a wide range of natural and agricultural habitats [START_REF] Ball | Potentilla L[END_REF][START_REF] Delgado | Karyosystematic study of Potentilla L. subgen. Potentilla (Rosaceae) in the Iberian Peninsula[END_REF][START_REF] Lesica | Effects of prescribed fire and season of burn on recruitment of the invasive exotic plant, Potentilla recta, in a semiarid grassland[END_REF][START_REF] Perkins | Age structure and age-related perfomance of sulfur cinquefoil (Potentilla recta)[END_REF]. P. recta has been reported to contain 2-pyrone-4,6-dicarboxylic acid, which seems to be a chemotaxonomic marker for the Potentilla species [START_REF] Wilkes | Isolation, characterization, and systematic significance of 2-pyrone-4,6-dicarboxylic acid in Rosaceae[END_REF]. Ranges of seasonal changes in the content of chlorophyll, carotenoids, free organic, ascorbic and triterpenic acids, neutral triterpenoids and fat oils in some organs and overground phytomass of P. recta in the conditions of Belarus have also been determined [START_REF] Rupasova | Seasonal accumulations of chloroplastic pigments, organic acids, and terpenoids in the plant Potentilla recta L. upon introduction in Belarus[END_REF]. Pharmacological studies of the plant have so far focused on preliminary antimicrobial activities, as well as the inhibitory action on h-AR, and haematological activity, including antiplatelet aggregating and anti-coagulating activities [START_REF] Enomoto | Inhibitory effects of traditional Turkish folk medicine on aldose reductase (AR) and haematological activity, and on AR inhibitory activity of quercetin-3-O-methyl ether isolated from Cistus laurifolius L[END_REF][START_REF] Tosun | Antimicrobial activity of some plants used in folk medicine in Turkey[END_REF][START_REF] Tomczyk | Antimicrobial activity of Potentilla species[END_REF].

Considering the traditional use of the Potentilla L. species, it would be meaningful to investigate its biological properties in the dental field. The purpose of this study was to evaluate the effects of extracts (aqueous, 50% ethanolic) and fractions prepared by the partitioning of aqueous methanolic extract with solvents of different polarities (diethyl ether, ethyl acetate and n-butanolic) from the aerial parts of P. recta against cariogenic Streptococcus spp. strains (S. mutans CAPM 6067, S. sobrinus CAPM 6070, S. sobrinus GCM 20381 and S. sobrinus/downei CCUG 21020).

Furthermore, their inhibitory effects on water-insoluble α-(1→3)-, α-(1→6)-linked glucan (mutan) and artificial dental plaque formation were examined. The phytochemical profile of active constituents in the investigated samples and plant material was also analyzed. Supernatans were filtered through a funnel with glass wool, which was washed with 5 mL of solvent and concentrated to dryness under vacuum (Büchi System, Switzerland) at controlled temperature (40 ± 2°C) and subjected to lyophilization using LABCONCO vacuum concentrator until a constant weight were obtained. Yields: PRE -82 mg (4.10%); PRE1 -96 mg (4.81%).

Preparation of Subfractions.

Accurately weighed 2.0 g quantities of plant material in powder form were separately extracted with methanol (3 x 50 mL) and once with 50 mL of 80% (v/v) methanol in an ultrasonicator bath (Sonic-5, POLSONIC, Poland) at controlled temperature (40 ± 2 °C) for given certain time 45 min. After solvent evaporation under reduced pressure from each sample, the methanolic extracts were diluted with water and successively partitioned between chloroform and then diethyl ether (PRE2), ethyl acetate (PRE3) and n-butanol (PRE4). All Switzerland) (temperature: 40 ± 2°C) and subjected to lyophilization using LABCONCO vacuum concentrator until a constant weight were obtained. Yields: PRE2 -13 mg (0.61%); PRE3 -46 mg (2.34%); PRE4 -93 mg (4.67%).

Phytochemical Profile

Determination of Total Phenolic Content. Total phenolic contents in extracts and subfractions (PRE-PRE4) were determinated spectrophotometrically at 765 nm (SPECORD 40, Analytik 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 [START_REF] Tawaha | Antioxidant activity and total phenolic content of selected Jordanian plant species[END_REF]. A 50 µL sample aliquot of extract and subfraction were dissolved in 450 µL of distilled water and 2.5 mL of 0.2 N Folin-Ciocalteu's reagent. After 5 min 2 mL of saturated sodium carbonate Na 2 CO 3 solution (75 g/L) were added. Samples were vortexed and incubating in the dark 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 I.

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 VI (2002). Stock solution was prepared from 1.0 g of the powdered sample mixed with 25 mL of water (two times) and shacked for 30 min each, then filtered. Phenolic acids were determined from 1 mL aliquot stock solution mixed with 5 mL of water, 1 mL hydrochloric acid (18g/L), 1 mL of Arnov's reagent and 1 mL of sodium hydroxide solution (40g/L) and diluted with water to 10 mL. Phenolic acids were measured

A × 1.7544/m,
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 II and are means of experiments conducted in triplicate.

Determination of Total Flavonoid Content. The total content of flavonoids was determined by the spectrophotometric method by [START_REF] Christ | Determination of the amount of flavonol derivatives in drugs[END_REF] and followed the procedure described in Polish Pharmacopoeia VI (2002). Each powdered sample (0.6 g) was mixed with 20 mL of acetone, 2 mL of 25% hydrochloric acid (281 g/L) and 1 mL of 0.5% urotropine (methenamine) solution (5 g/L) 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 20 mL of acetone. The extracts were mixed and diluted with acetone to 100 mL in a volumetric flask. Then, 20 mL of this solution was diluted with 20 mL of water, extracted with 15 mL of ethyl acetate and then, three time with 10 mL of ethyl acetate. Organic phases were mixed and washed twice with 40 mL of water, fitered to volumetric flask and diluted with ethyl acetate to 50 mL. To four volumetric flasks 10 mL of the obtained solution was added. Then, to three flasks 2 mL of aluminium chloride (20 g/L -methanolic solution) 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 aluminium chloride in a methanolethyl acetate-acetic acid medium. The contents of total flavonoids, expressed as quercetin or hyperoside equivalent on dry weight, were calculated from the expression, respectively: II.

A × 0.
Determination of Total Proanthocyanidin Content. The total proantocyanidin content was measured according to the European Pharmacopoeia 6 th (2007). Accurately weighted 2.5 g of plant material was heated under reflux for 30 min with 30 mL of ethanol (70% v/v). After that extract was filtered and the residue was flashed with 10 mL of ethanol (70% v/v). Then 15 mL of 25% hydrochloric acid was added with 10 mL of water. 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 50 mL od solution was evaporated to about 3 mL and transferred to a separating funnel with 15 mL of water.

The solution were 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 from the expression: A × 500/75 x m, 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 II.

Anticariogenic Activity

Bacterial strain, media and growth conditions. The cariogenic streptococci used in this of Göteborg, Sweden). The strains were stored as glycerol stocks at -20°C. Bacteria were grown at 37°C in minimum medium for S. mutans [START_REF] Fujiwara | Development of a minimal medium for Streptococcus mutans[END_REF] containing (g/L) glucose (10), Lglutamic acid (2), L-cysteine-HCl (0.2), L-leucine (0.9), NH 4 Cl (1), K 2 HPO 4 (3.5), KH 2 PO 4 (1.5), NaHCO 3 (4.2), MgSO 4 •7H 2 O (1.2), MnCl 4 •4H 2 O (0.02), FeSO 4 •7H 2 O (0.02), sodium pyruvate (0.6), and (mg/L) riboflavin (1), thiamine-HCl (0.5), biotin (0.1), nicotinic acid (1), p-aminobenzoic acid (0.1), calcium pantothenate (0.5), pyridoxal-HCl (1), tris-maleate buffer (pH 7.4) to 1 L. The final pH of the medium was 7.1. After 24 h incubation bacterial cultures were centrifuged (17000 x g for 30 min), and the supernatants were used as cell-free glucosyltransferases.

Agar-well diffusion method. For the investigation of the antibacterial activity the agar-well diffusion method was used. Samples for the tests were dissolved in dimethylsulfoxide (DMSO).

Wells (5 mm diameters) were cut in the inoculated agar plates. The solutions of tested extracts (50 µL) were spotted onto such prepared plates (5 mg of preparations per well) and diameter of inhibition zone was measured after 24 h incubation at 37°C. Antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by the plant extract.

Inhibition of mutan synthesis. The inhibitory effect of the P. recta extracts and subfractions on mutan synthesis was examined. A two-fold serial dilution of each extract (concentration ranging from 6.25 to 800 µg/mL) in 1% DMSO (100 µL final volume) was prepared in Eppendorf tubes.

All tubes were filled with 1.0 mL of 24-h-old post-culture supernatant of the tested bacterium and 100 µL of sucrose solution (final concentration of 3%, w/v) in 0.1 M potassium phosphate buffer (pH 6.0) in the presence of 0.05% sodium azide as a preservative. Ellagic acid (Sigma-Aldrich, Germany), in form of sodium ellagate, from 6.25 to 1.87 µg/mL, was used as the positive control, the medium without extracts was used as the non-treated control. After incubation for 24 h at 37°C, the formed water-insoluble polysaccharide (mutan) was collected by centrifugation at 16099 x g for F o r P e e r R e v i e w 9 10 min, washed thoroughly with deionized water and determined, utilizing phenol-sulfuric acid method with glucose as a standard [START_REF] Dubois | Colorimetric method for determination of sugar and related substances[END_REF].

Inhibition of biofilm formation. The effect of P. recta crude extracts and subfractions on cariogenic streptococci biofilm formation was examined. A two-fold serial dilution of each extract in 1% DMSO (final volume 300 µL) was prepared in glass tubes (1.2 × 9.5 cm). All tubes were filled up with 1.2 mL of culture medium with sucrose (final concentration of 3%, w/v) and then inoculated of 20 µL of overnight broth culture of the tested bacterium. Final concentrations of extracts and subfractions ranged from 6.25 to 800 µg/mL. Ellagic acid (in form of sodium ellagate, from 1.87 to 50 µg/mL) was used as the positive control, the medium without extracts was used as the non-treated control. After incubation for 24 h at 37°C, media from each tube were decanted and planktonic cells were removed by washing with distilled water. The adhered biofilm was stained with 1.5 mL of 1% erythrosin B for 5 min, rinsed thoroughly with water, and dried at room temperature. The bound dye was then removed from biofilm with 3 mL of 1M NaOH. Tubes were set on a shaker for 5 min to allow full release of the dye. Biofilm formation was quantified by measuring optical density at 525 nm. Data analysis. Statistical analysis was performed on all the three replicates from each treatment.

Standard deviations and analysis of variance (ANOVA) were determined using and analysis of variance (ANOVA). Significance was evaluated at p < 0.05 for all tests. Mean separation was accomplished using least significant difference test.

RESULTS AND DISSCUSION

Over the past years many studies on the anticariogenic effect of polyphenols extracted from different types of plants (such as tea, apple or hop) have been reported [START_REF] Ito | Flavonoids as a possibile preventive of dental caries[END_REF][START_REF] Tagashira | Inhibition by hop bract polyphenols of cellular adherence and water-insoluble glucan synthesis of mutans streptococci[END_REF][START_REF] Sakanaka | Antibacterial substances in Japanese green tea extract against Streptococcus mutans, a cariogenic bacterium[END_REF][START_REF] Yanagida | Inhibitory effects of apple polyphenols and related compounds on cariogenic factors of mutans streptococci[END_REF]. These earlier investigations proved that plant polyphenolics have a wide range of pharmacological properties, such as anticariogenic activity in many pharmacological models such as in vitro, in vivo and studies on humans [START_REF] Petti | Polyphenols, oral health and disease: A review[END_REF]. The development of therapeutic agents aimed at disrupting both colonization of the teeth by dental pathogens and the subsequent formation of dental plaque is one of the prime strategies to reduce the incidence of tooth decay [START_REF] Bowen | Wither or whither caries research?[END_REF]. Among the different targets, glucosyltransferases (GTFs) produced by cariogenic streptococci such as S. mutans and S. sobrinus play a crucial role as one of the most important virulence factors since they are involved in both the formation of the plaque matrix and colonization of oral bacteria. Mutans streptococci produce at least three different GTFs: GTF B, which synthesizes mostly insoluble α-(1→3)-linked glucan; GTF C, which synthesizes a mixture of insoluble and soluble α-(1→6)-linked glucan, and GTF D, which synthesizes soluble glucan. Of these, GTFs B and C appear to be the most strongly associated with dental caries [START_REF] Duarte | Effect of a novel type of propolis and its chemical fractions on glucosyltransferases and on growth and adherence of mutans streptococci[END_REF]. The GTFs synthesize up to 30-40% (dry weight) of the polysaccharide fraction of the dental plaque matrix [START_REF] Shani | In vitro assessment of the antimicrobial activity of a local sustained release device containing amine fluoride for the treatment of oral infectious diseases[END_REF]. These glucans (especially water-insoluble mutan) can enhance the pathogenic potential of dental plaque by promoting the accumulation of oral bacteria on human teeth. Therefore, one of the strategies to control plaque formation is to inhibit the oral pathogens' growth and the activity of glucosyltransferases.

The antimicrobial activities of P. recta extracts against mutans streptococci were assessed by the presence or absence of inhibition zones and zone diameter (Table 3). The results showed that extracts and subfractions, except PRE against S. mutans 6067, inhibited the growth of all the tested 4 and5). The action of extracts on water-insoluble mutan synthesis by cell-free glucosyltransferase preparations from each tested bacteria was expressed as the relative amount of mutan produced in the presence of fractions from P. recta compared to the amount produced in the absence of any extracts. The extracts showed the following order of potency: PRE3>PRE4>PRE1>PRE2>PRE.

The most effective was ethyl acetate subfraction (PRE3). The minimum mutan inhibition concentrations (MMIC 50 ) of preparations ranged from 6.25 to 50 µg/mL for all tested strains (Table 4). The data obtained in this study showed that glucosyltransferases of S. sobrinus 20381 were more resistant to the influence of extracts than other GTF preparations. In the case of biofilm formation, the MBIC 50 values of agents ranged from 25 to 400 µg/mL (Table 5), and the order of potency was PRE3>PRE1>PRE4>PRE>PRE2. S. sobrinus/downei 21020 biofilm demonstrated the lowest susceptibility for all extracts and subfractions compared with biofilms formed by other strains.

Similar, the same ethyl acetate subfraction PRE3 significantly reduced both mutan synthesis and biofilm formation at the concentration of 12.5 and 100 µg/mL, respectively (p < 0.05). That suggests a high concentrations of active polyphenolic constituents with anticariogenic activity in the investigated subfractions PRE3 (Table 1). The inhibitory effects of all analysed Potentilla extracts were significantly greater than those previously observed in other natural products, for instance Nidus Vespae extracts (MBIC 50 from 8 to 64 mg/mL) [START_REF] Xiao | Effects of Nidus Vespae extract and chemical fractions on adherence and biofilm formation of Streptococcus mutans[END_REF].

The precise bioactive molecules from P. recta extracts and fractions that mediate the inhibitory effects against the viability of oral pathogenic bacteria are still unknown. Based on the preliminary phytochemical profile in the present study, the activity of these extracts and fractions might be related to the presence in plant material of complex polyphenolic compounds like phenolic acids, tannins (hydrolyzable tannins and proanthocyanidins), as well as flavonoids. But in many The amount of total polyphenolic compounds (phenolic acids, tannins, flavonoids, proanthocyanidins) in investigated plant extracts and subfractions in most cases correlated with their anticariogenic activity. The results obtained in the present study shows that all the analyzed fractions and extracts have relatively high concentrations of polyphenolic compounds. However, all the fractions and extracts had different total polyphenol content (p < 0.05). The ethyl acetate fraction showed the highest values and diethyl ether fractions showed the lowest one (Table 1). The content of total polyphenols ranged from 20.9 ± 0.5 mg GAE/g dw for PRE2 to 33.6 ± 1.3mg GAE/g dw for PRE3. All these differences in the values of total phenolic content in all the analyzed fractions and extracts can be attributed to the differences in the composition of these fractions and extracts. It was obvious that the total phenolic compounds determined by Folin-Ciocalteu's method had not given a full characterisation of the quality and quantity of the various groups of polyphenolic compounds. Chromatographic analysis of the ethyl acetate fraction (PRE3) indicated the presence of, in particular, hydrolysed (ellagitannins or gallotannins) and condensed tannins (proanthocyanidins), as well as glycosylated derivatives of flavonoid compounds (2D-TLC analysis, data not shown). In contrast, the chromatographic analysis of diethyl ether fractions proved the highest concentrations of phenolic acids, flavonoid aglycones and acylated flavonoids (2D-TLC analysis, data not shown). The presence of these groups of compounds has encouraged us to determine them in plant material by weight and spectrophotometrical methods. The results (Table 2) obtained showed that aerial parts of P. recta had relatively very high concentrations of tannins (101.0 ± 1.7 mg/g dw) proanthocyanidins (3.7 ± 0.6 mg/g dw) and phenolic acids (5.4 ± 0.3 mg/g dw), as well as flavonoids (2.7 ± 0.2 mg/g dw) and (3.9 ± 0.4 mg/g dw), calculated as quercetin, flavonol type compound and parallel as a glycoside hyperoside, respectively. Summarizing, the data presented here imply that the anticariogenic activity of P. recta extracts and fractions was much stronger than that of those reported for other Potentilla spp.

Recently, [START_REF] Wiater | Anticariogenic activity of the crude ethanolic extract of Potentilla erecta (L.) Raeusch[END_REF] reported that ethanol extract of rhizomes of P. erecta inhibited both total GTF activity and mutan synthesis in the range of 500 -2000 µg extract/mL. From these comparative data, it can be concluded that different extracts of P. recta could be useful for the development of new anticariogenic agents in a wide range of oral care products such as toothpastes, mouthwashes and chewing gum. Most certainly, the polyphenolic compounds are responsible for this kind of pharmacological property. Regardless of the obtained results, there is a need for further investigation directed towards the isolation and identification of active compounds in the extracts and subfractions of the aerial parts of P. recta as well as determination of their influence on the examined activities.

  Seeds of P. recta were requested from the Giardino Botanico Alpino, Cogne, Italy (index seminum: 1549). Plants were cultivated in a common plots at the Medicinal Plants Garden near Medical University of Białystok, Poland. Aerial parts of plants were collected during June-August 2007-2009. Voucher specimens of plants (No. PRE-06019) have been deposited in the Herbarium of the Department of Pharmacognosy, Medical University of Białystok, Poland. Preparation of Extracts and Subfractions. Different solvent systems (water, 50% ethanol, diethyl ether, ethyl acetate and n-butanol) were used to prepared the extracts and subfractions. Preparation of Extracts. Powdered plant material (2.0 g) was separately extracted with water (2 x 150 mL) -PRE or 50% ethanol (2 x 150 mL) -PRE1 in an ultrasonicator bath (Sonic-5, POLSONIC, Poland) at controlled temperature (40 ± 2°C) for given certain time 45 min.

  to dryness under vacuum controlled temperature (Büchi System,

  nm The percentage of phenolic acids, expressed as caffeic acid equivalent on dry weight, is calculated from the expression:

  875/b and A ×1.25/b, 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 TableII.

  Tannin Content. The total tannin content was determined by the weight method with hide powder according to the DAB 10 (1998). The results are given in Table

  study included Streptococcus mutans CAPM 6067, S. sobrinus CAPM 6070 (The Collection of Animal Pathogenic Microorganisms, Brno, Czech Republic), S. sobrinus GCM 20381 (formerly S. mutans 20381) (The German Collection of Microorganisms, Braunschweig, Germany) and S. sobrinus/downei CCUG 21020 (formerly S. mutans OMZ 176) (The Culture Collection, University

  Maximal inhibition zones, in the range of 18 to 24 mm, were obtained for the PRE2 fraction. All five P. recta preparations exhibited an inhibitory effect on mutan and oral streptococci biofilm formation in a concentration-dependent manner (Figures 1, 2 and Tables

  effects can not be attributed solely to polyphenolics, because plant extracts contain several other phytochemicals.

Figure 1 .

 1 Figure 1. The inhibitory effects of all investigated P. recta preparations (PRE-PRE4) on mutan

Figure 2 .

 2 Figure 2. The inhibitory effects of all investigated P. recta preparations (PRE-PRE4) on mutans

F

  Figure 1

Table 1 .

 1 Total phenolic content in the obtained extracts and subfractions from P. recta All data are shown as means ± SD of at least three independent experiments, each performed in duplicated samples (p < 0.05).

	Extracts	Total phenolic content
	(mg GAE/g dry weight) a
	PRE	23.8 ± 0.7
	PRE1	27.4 ± 0.6
	PRE2	20.9 ± 0.5
	PRE3	33.6 ± 1.3
	r o PRE4	25.4 ± 0.9
	P
	e
	e r
		R
		e
		v i e
		w
		18

a

Table 2 .

 2 Content of polyphenolic compounds in P. rectaPolyphenols content (mg/g dry weight) a Results are means ± SD of three different experiments.

	Phenolic acids	Flavonoids	Tannins	Proanthocyanidins
		Quercetin	Hyperoside
	5.4 ± 0.3	2.7 ± 0.2	3.9 ± 0.4	101.0 ± 1.7	3.7 ± 0.6

a

Table 3 .

 3 Effects of P. recta extracts and subfractions on bacterial viability obtained by agar-well diffusion method a Antibacterial activity is expressed as the diameter of inhibition zone, including the well diameter (5 mm), DMSO was used as negative control. ni -no inhibition * p < 0.05, ** p < 0.01, *** p < 0.001

		Phytotherapy Research	Page 20 of 24
	Samples/control		Zone of inhibition (mm) a
		S. sobrinus	S. sobrinus	S. mutans	S. sobrinus/downei
		20381	6070	6067	21020
	PRE	16**	10	ni	16***
	PRE1	12***	15**	12	17***
	PRE2	24*	18***	18***	21*
	PRE3	14***	14*	16**	21*
	PRE4	11***	11	10	33***
	Streptomycin	21	11	11	24
	250 µg/ml

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Table 4 .

 4 Effects of P. recta extracts and subfractions on the mutan synthesis MMIC 50 -minimum mutan inhibition concentration, the lowest agent concentration that showed > 50% inhibition on mutan synthesis.

	MMIC 50 (µg/mL)
	Strains
	PRE PRE1 PRE2 PRE3 PRE4

Table 5 .

 5 Effects of P. recta extracts and subfractions on the formation of mutans streptococci biofilm MBIC 50 -minimum biofilm inhibition concentration, the lowest agent concentration that showed > 50% inhibition on biofilm formation.

			MBIC 50 (µg/mL)
	Strain
		PRE PRE1 PRE2 PRE3 PRE4
	S. sobrinus 20381	50	50	200	25	50
	S. sobrinus 6070	200 100	400	100	100
	S. mutans 6067	200 100	200	50	200
	S. sobrinus/downei 21020 200 100	200	200	400

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Acknowledgements: This study is financially supported by the Polish Ministry of Science and Higher Education (Grant No. N N405 621638)