In vitro study of antioxidant capacity and antibacterial activity on oral anaerobesof a grape seed extract

Aurelie Furiga

, Aline Lonvaud-Funel

, Cecile Badet

a,b,

Laboratoire Odontologique de Recherche, UFR d’Odontologie, Université Victor Segalen Bordeaux 2, 16 Cours de la Marne, 33082 Bordeaux Cedex, France

UMR 1219



nologie, Université Victor Segalen Bordeaux 2, INRA, ISVV, 351 cours de la Libération, 33405 Talence, France

a r t i c l e i n f o

Article history:

Received 13 May 2008Received in revised form 9 July 2008Accepted 21 August 2008

Keywords:

Grape seed extractOral anaerobesAntibacterial activityAntioxidant

a b s t r a c t

Grape seeds are considered rich sources of polyphenolic compounds that showantioxidant or antimicro-bialeffects. Theaimofthepresent workwastoinvestigatetheeffectofagrapeseedextract (GSE)ontwooral anaerobes closely associated with periodontal diseases and its antioxidant action.Theantimicrobial activitywas determinedusingthemacrodilutionbrothtechnique andalsotestedonamulti-species bioﬁlmgrownonsaliva-coatedhydroxyapatitediscs. Theevaluationof antioxidant activ-ity was based on the capacity of a sample to scavenge the ABTS radical cation as compared to a standardantioxidant(Trolox).GSEhadabacteriostaticeffectontheanaerobes.Ataconcentrationof2000

g/ml,itsignificantlydecreasedtheformationofbiofilm.HighTEACvalueswereregisteredandthisextractexhib-ited greater antioxidant capacity than vitamins C and E.These findings indicated that GSE could be used in oral hygiene for the prevention of periodontitis.

1. Introduction

Periodontitis and other acute periodontal diseases are dentalplaque mediated (Page & Kornman, 1997). Among the numerousspecies embedded in the plaque bioﬁlm,

Porphyromonas gingivalis

and

Fusobacterium nucleatum

are closely associated with variousforms of these diseases (Moore & Moore, 1994; Ximénez-Fyvie,Haffajee, & Socransky, 2000). If

F. nucleatum

is the most abundantGram-negative anaerobe in the sub-gingival plaque and plays animportantrole initsformation,

P. gingivalis

isalsoamajor putativepathogenic bacteriumthat occurs in severe adult periodontitis andacute periodontal diseases (Bradshaw, Marsh, Watson, & Allison,1998; Slots, 1999).The preventionof oral diseases ismainlytargetedat thecontrolof dental plaque (Baehni & Takeuchi, 2003). Mechanical removal isnot always sufﬁcient and the use of chemical antiplaque agents,particularlypolyphenoliccompoundsthat havepreviouslyshowedanti-adhesiveeffects,isoftenhelpfuland(Dagliaetal.,2002;Ham-ilton-Miller, 2001; Marsh & Bradshaw, 1993; Sakagami, Oi, & Sa-toh, 1999; Xiao et al., 2007).Besides dental plaque deposition, another mechanism can beimplicated in periodontal diseases development. Some studieshave reported that the excess production of reactive oxygen spe-cies (ROS) leads to damages of gingival tissues, periodontal liga-ment and alveolar bone (Batista, Silva, Chun, & Lara, 2002;Battino, Bullon, Wilson, & Newman, 1999; Canakci, Cicek, & Cana-kci,2005;Waddington,Moseley,&Embery,2000;Tsaietal.,2005).The search is therefore on for an antioxidant that could be used tocontrolthesediseases,andpolyphenoliccompoundsarelikelycan-didates (Houde, Grenier, & Chandad, 2006).Grape (

Vitis vinifera

) seeds are considered rich sources of poly-phenoliccompounds,mainlymonomericcatechinandepicatechin,gallic acid, and polymeric and oligomeric procyanidins (Monagas,Gómez-Cordovés, Bartolomé, Laureano, & Ricardo da Silva, 2003).These polyphenolic compounds showed various biological effects,such as antioxidant or antimicrobial (Baydar, Sagdic, Ozkan, &Cetin, 2006; Jayaprakasha, Selvi, &Sakariah, 2003). However, thereislimitedresearchontheiractiononoral diseases (Sakagamiet al.,1999; Smullen, Koutsou, Foster, Zumbé, & Storey, 2007).Thepresentworkinvestigatedtheactionofagrapeseedextractthat could be used in prevention or treatment of periodontal dis-eases. For this, we examined its antibacterial effect on planktoniccells of

F. nucleatum

and

P. gingivalis,

its antiplaque activity on amultispecies bioﬁlm and its antioxidant capacity.It is, to our knowledge, the ﬁrst paper which investigates bothantibacterial and antioxidant functions of this extract for oral hy-giene purposes.

2. Materials and methods

2.1. Plant material

Grape seed extract (GSE) was extracted fromthe vine

V. vinifera

by Societe Française de Distillerie (Vallon Pont d’Arc, France). It

0308-8146/$ - see front matter

Corresponding author. Address: Laboratoire Odontologique de Recherche, UFR d’Odontologie, Université Victor Segalen Bordeaux 2, 16 cours de la Marne, 33082,Bordeaux Cedex, France. Tel.: +33 5 57 57 30 07; fax: +33 5 57 57 30 10.

E-mail address:

cecile.badet@odonto.u-bordeaux2.fr(C. Badet).Food Chemistry 113 (2009) 1037–1040

Contents lists available atScienceDirect

Food Chemistry

journal homepage:www.elsevier.com/locate/foodchem

contains 97% (w/w) polyphenols, mainly catechin and epicatechin(polyphenolic contents determined by the supplier).

2.2. Bacterial strains and growth conditions

The two anaerobe micro-organisms used in this study were

P. gingivalis

ATCC 33277 and

F. nucleatum

ATCC 10953. For the mul-ti-species bioﬁlm formation, six strains were used:

Streptococcusmutans

ATCC25175,

Streptococcus sobrinus

ATCC33478,

Lactobacil-lus rhamnosus

ATCC 7469,

Actinomyces viscosus

ATCC 15987 andthe two anaerobes.All strains were cryo-preserved at

Actinomyces

and

Streptococcus

wereculturedin trypticsoy broth(Difco, St. Sauveur,France), the lactobacilli in MRS medium (Merck, Darmstadt, Ger-many), andtheanaerobesinWilkinsChalgrenanaerobebroth(Ox-oid, Dardilly, France).

2.3. Antimicrobial activity

TheantimicrobialactivityofGSEforthetwooralanaerobeswasexamined by determining the minimal inhibitory concentration(MIC) and minimal bactericidal concentration (MBC) using themacro dilution broth technique.Brieﬂy, an overnight culture of approximately 5

CFU/mlwas inoculated into tubes containing test compound dilutionsand incubatedat 37

C for 24h. The MIC was deﬁned as the lowestconcentrationof test compoundableto restrictbacterial growthtoa level lower than 0.05 at 650nm.For MBC determination, an aliquot (50

l) from tubes contain-ing no visible growth was diluted 100-fold in ultra-pure water,and then sub-cultured onto Wilkins Chalgren anaerobe agar sup-plementedwithbloodandGNsupplement(Oxoid).Theplateswereincubatedanaerobicallyat37

Cfor48–72h.TheMBCwasdeﬁnedas the lowest concentration of test compounds that did not permitany visible growth after the incubation period (99.9% killed).Each concentration of the extract was tested in triplicate. Thevehicle (ultra-pure water) was used as negative control.

2.4. Inhibition of bioﬁlm formation: Antiplaque activity 2.4.1. Bioﬁlm assay

The biofilm was assayed according to the model described byGuggenheim, Giertsen, Schupbach, and Shapiro (2001). Only fewmodifications have been implanted, mainly changes in bacterialcomposition. Briefly, bacterial inoculum was prepared by combin-ing 1ml of overnight pre-cultures of each previously cited species(beforehand adjusted to an optical density of 1.0±0.02 at 550nm)in fluid universal medium (FUM). Biofilms were developed onhydroxyapatite (HA) discs (Clarkson Chromatography ProductsInc, USA) coated with human pasteurised saliva (30min at 65

C).Thesterile24-wellcellcultureplate,containingineachwell,anHA-coated disc and a mixture of saliva (800

l), FUM (800

l, con-taining 0.15% (w/v) glucose and 0.15% (w/v) sucrose) and bacterialinoculum(200

l), wasincubatedanaerobicallyat 37

C. After16hincubation, HA discs were exposed for 1min to the test compoundatvariousconcentrationsorultrapurewater(control).Afterrinsing,they were replaced in their wells. This test compound treatmentwas repeated after 4h and 8h. Between each exposure, the platewas incubated anaerobically at 37

C. After 16h incubation, discswereexposedagainthreetimestotestscompoundsat4hintervals.

2.4.2. Harvesting the bioﬁlm

At the end of the experiment (age of bioﬁlm of 64h), HA discswere washed with physiological saline to remove poorly-adherentbacteria. Toharvestadherentcells,eachdiscwasplacedinasterileplasticPetridish,anddiscsurfaceswerescrapedwithasterileden-tal root curette. The surface of the scraped disc and the petri platewere rinsed with physiological saline (1000

l). Aliquots of har-vested bioﬁlm were diluted and spiral-plated onto Mitis Salivariusagar+tellurite (Difco) for

Streptococcus

, MRS agar (Merck) for

Lac-tobacillus,

trypticase soy agar (Difco) for

Actinomyces

, or WilkinsandChalgrenanaerobeagarsupplementedwithbloodandGNsup-plement (Oxoid) for

Fusobacterium

and

Porphyromonas

. Agar plateswere incubated anaerobically at 37

C for 48–72h. Gram stainingwas performed in order to conﬁrm the identity of species on eachmedium.The CFU per population for triplicate discs inoculated with dif-ferent concentrations of grape seed extract (0–2000

g/ml) wereaveraged and subjected to logarithmic transformation.

2.5. Antioxidant activity: ABTS assay

All chemical reagents were purchased from Sigma–Aldrich(Saint–Quentin Fallavier, France).ThemethodisbasedonthecapacityofasampletoscavengetheABTSradicalcation(ABTS

),ascomparedtoastandardantioxidant(Trolox). ABTS

was generated from ABTS as previously described(Reetal.,1999).Brieﬂy,theABTS

solutionwasproducedbyreact-ing 7mM ABTS stock solution with 2.45mM potassium persulfate(ﬁnal concentration) and allowing the mixture to stand in the darkat room temperature for 12–16h. Prior to use in the assay, theABTS

solution was diluted with PBS to an absorbance of 0.70(±0.02) at 734nm and equilibrated at 30

C. Grape seed extract,ascorbicacid(ascontrol)andTrolox(asstandard)(20

l)weredis-solved in PBS, and then added to the ABTS

solution(1980

l). Theabsorbance reading was taken exactly 6min after initial mixing.Appropriate solvent blanks were run in each assay.Three different dilutions of the compound under investigationwere selected which produced absorbance values in the most lin-ear region of the Trolox dose–response curve (20–80% inhibitionof the blank value). All determinations were carried out at leastthree times and the three dilutions were analysed in triplicate.The dose–response curves obtained with the antioxidant mixturesand Trolox were plotted as the percentage of absorbance decreaseagainst the amount of antioxidants expressed as

g/ml (samples)or in micromolar units (Trolox). The Trolox equivalent antioxidantcapacity (TEAC) was calculated as the ratio between the slopes of the dose–response curves of the samples and Trolox.

2.6. Statistical analysis

Student

-test was used to calculate the significance of the dif-ference between the mean effects of a given compound comparedwiththecontrolgroup. Statisticallysignificantvaluesweredefinedas

<0.05.

3. Results

3.1. Antimicrobial activity

The antibacterial activity of the test compound against the bac-terial strains shows high values of MIC (Table 1). The MBC was

Table 1

Antibacterial activity of grape seed extract (GSE)

MIC (

g/ml) MBC (

g/ml)

gingivalis

4000 8000

nucleatum

2000 8000Minimum inhibitory concentration (MIC) and minimum bactericidal concentration(MBC) after 24h incubation with bacteria.1038

A. Furiga et al./Food Chemistry 113 (2009) 1037–1040

found to be 2 (for

P. gingivalis

) to 4 (for

F. nucleatum

) times higherthan MIC values.

3.2. Inhibitory effect on bioﬁlm

The inhibitory effects of GSE on bioﬁlm formation are dose-dependent (Fig. 1). At the concentration of 2000

g/ml (sub-MBClevels), GSE showed its best activity, even if the effect is differentaccordingtothebacteriacomposingthebioﬁlm.Higherconcentra-tions in this extract led to a fall of its effectiveness, principallycaused by its poor dissolution in water.

3.3. Antioxidant activity

The antioxidant capacity of chlorhexidine and GSE is shown inTable 2. Since TEAC is a quantiﬁcation of the effective antioxidantactivity of the extract, a higher TEAC would imply greater antioxi-dant activity of the samples. GSE presented a high TEAC value,proving its capacity to scavenge the ABTS

radical cation.

4. Discussion

In order to overcome the side effects of antiseptics like chlorh-exidine (e.g., teeth staining), previous works have investigated theeffects of polyphenolic compounds, mainly extracted from teas orcranberries, for anti-cariogenic or anti-periodontitis purpose withquite interesting results (Hamilton-Miller, 2001; Labrecque, Bodet,Chandad, & Grenier, 2006; Matsumoto, Minami, Sasaki, Sobue, &Hamada, 1999; Steinberg, Feldman, Ofek, & Weiss, 2004). In thiscontext, we have focused on the study of biological capacities of a grape seed extract, known for its content of active polyphenols(Baydar et al., 2006; Jayaprakasha et al., 2003). Indeed, to ourknowledge, only a few studies have been carried out to examinetheimplicationofwineandgrapeextractsinthepreventionoforaldiseases (Houde et al., 2006; Smullen et al., 2007; Toukairin, Uch-ino, Iwamoto, Murakami, et al., 1991).The ﬁrst step of our experiment was the investigation of theantibacterial effect on bacteria implicated in periodontal diseases.According to results obtained with other bacterial species (Baydaretal., 2006;Houdeet al., 2006; Smullen,Koutsou,Foster, Zumbe, &Storey, 2007), GSE showed interesting effects, although the MICvalues are much higher than those obtained byToukairin et al.(1991),withseedsandskinofwinegrapeextracts.Onlyafewstud-ies have tested the effect of other polyphenolic extracts on anaer-obic bacteria implicated in periodontitis.Labrecque et al. (2006)reported that the growth and viability of

P. gingivalis

were unaf-fected by a non-dialysable material (NDM) prepared from cran-berry juice. On the other hand, propolis extracts, containingpolyphenolic compounds like ﬂavonoids, showed an antibacterialactivity against

F. nucleatum

and

P. gingivalis

(Koo et al., 2000;Boyanova, Kolarov, Gergova, & Mitov, 2006; Yamamoto & Ogawa,2002).Dental plaque, implicated in oral diseases, is a very complexbiofilm which gives to bacteria a protection against antimicrobialagents (Gilbert, Das, & Foley, 1997; Mah & O’Toole, 2001; Wilson,1996). So another important step of our investigation was to eval-uate the activity of our polyphenolic extract on an experimentalmulti-species biofilm validated by various studies (Guggenheimet al., 2001; Shapiro, Giertsen, & Guggenheim, 2002). Only fewworkshavebeencarriedoutonsuchbiofilms.However,ourresultscan be related to those of Shapiro et al. (2002), who found thatmouth rinses containing plant extracts were effective but less sothan those containing chlorhexidine.Periodontitisisinitiatedbythesub-gingivalbiofilmbutthepro-gression of destructive disease appears to be dependent upon anabnormalhostresponse(Page&Kornman,1997).Excessofreactiveoxygen species release is implicated in the inflammatory process(Gustafsson & Asman, 1996). So, evidence is emerging to implicateoxidative stress in the pathogenesis of periodontitis (Chapple &Matthews, 2007). Hence, it could be of interest to use specific anti-oxidant nutrients in the field of oral disease prevention.Fewstudieshavebeencarriedoutonantioxidantactivityoforalhygiene products. Using the same test as us,Battino et al. (1999)showed that among 12 differently antioxidant-enriched tooth-pastes,onlythosecontainingsodiumascorbylphosphatedisplayedclearantioxidant activity. Withanothertype of experiment,Houde

Fig. 1.

Effect ofdifferentconcentrationsofgrapeseedextract (GSE)onbacteriacomposingthemulti-speciesbioﬁlm. Resultsareexpressedasmeansandstandarddeviationsof triplicate experiments. Statistical differences (*,

<0.05;**,

<0.01 and***,

<0.001) between test compound and control (

=3).

Table 2

Antioxidant activity of grape seed extract (GSE)

Compounds TEACAscorbic acid 5.73±0.06Chlorhexidine 0.02±0.03GSE 7.01±0.18The Trolox equivalent antioxidant capacity (TEAC) corresponds to the micromolarconcentration of Trolox equivalent to a 1

g/ml solution of sample (the higher themore efﬁcient). Each value corresponds to the mean and standard deviation of thetriplicate of three separate concentrations within the linear interval (

=3).

A. Furiga et al./Food Chemistry 113 (2009) 1037–1040

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