Abstract

In the antimicrobial therapy of denture stomatitis, itis desirable to inhibit the growth of not only theprimary causative organism, Candida albicans, butalso other oral bacteria closely associated with thecondition. Three synthetic anti-candidal chalconeswere characterized and compared for theiradditional activity in inhibiting these causativebacteria. Among the tested chalcones, 2,4,2’-trihydroxy-5’-methylchalcone showed the highestactivity for different Gram-positive bacteria. Itinhibited the growth of streptococci, staphylococciand lactobacilli at 25.0-50.0 µg/mL which was lowerthan or comparable to its minimum inhibitoryconcentration for candida. It functioned with abactericidal action and leaked 260 nm-absorbingsubstances from the streptococcal cells. The anti-microbial activity of 2,4,2’-trihydroxy-5’-methyl-chalcone against both primary and secondarycausative agents suggests it could be useful as apotent therapeutic agent in denture stomatitis.

Key words: Oral bacteria, growth inhibition, anti-candidalchalcone, denture stomatitis.

(Received for publication July 1994. Revised December1994. Accepted January 1995.)

Introduction

Candida albicans is the most important aetiologi c a lagent for chronic atrophic candidosis (denturestomatitis), which develops in at least 50 per cent ofdenture wearers.1,2 In the treatment and prophylaxis

Australian Dental Journal 1997;42:(5):343-6

Growth inhibition of oral bacteria related to denturestomatitis by anti-candidal chalcones

Masaru Sato, PhD, DDS*

Hironori Tsuchiya, PhD†

Mioko Akagiri, DDS†

Nobuhiko Takagi, PhD, DDS†

Munekazu Iinuma, PhD‡

of denture stomatitis, antimicrobial plant-derivedcompounds (phytochemicals) have been studied aspossible therapeutic alternatives to antibiotics withu n avoidable side effects.3 - 5 Recently the anti-candidalactivity of phytochemicals with a chalcone skeleton(1,3-diphenyl-2-propene-1-one) was studied and itwas found that certain chalcones inhibited thegrowth of C. albicans.6 Such activity was furtherenhanced by structurally modifying them tohydroxyl derivatives.7

In addition to the primary causative agent, C.albicans, other oral bacteria also participate in thep athogenesis of denture stomat i t i s.8 In this condition,they possibly enhance fungal attachment to denturefitting surfaces v i a c o a g gr e g ation, increase thev i rulence of microflora on dentures by synergi s t i c a l l yinteracting with the fungi and promote fungalinvasiveness of mucosal tissues by damaging theepithelial cells. Therefore, it is desirable for anti-fungal agents additionally to possess a gr ow t hinhibitory effect on the secondary causative bacteria.The anti-bacterial activity of three anti-candidalchalcones against oral bacteria related to denturestomatitis were studied.

Materials and methods

Preparation of chalcones

Chemical structures of the chalcones used in thisstudy are shown in Fig. 1; 2,4,2’-trihydroxy-5’-m e t hylchalcone (THMC) and 2,4,2’-tri hy d r ox y-chalcone (THC) were synthesized based on amethod reported previously.7 In brief for THMC,2 ’ - hy d r ox y - 5 ’ - m e t hylacetophenone and diisopro-poxybenzaldehyde were reacted overnight at roomtemperature in an alkaline medium. The product,2’-hydroxy-2,4-diisopropoxy-5’-methylchalcone wastreated with BCl3 at room temperature for two

Australian Dental Journal 1997;42:5. 343

* D e p a rtment of Oral Microbiology, Asahi Unive rsity School ofD e n t i s t ry, Ja p a n .†Department of Dental Pharmacology, Asahi University School ofDentistry, Japan.‡Department of Pharmacognocy, Gifu Pharmaceutical University,Japan.

Since THMC showed the highest activity againsttested oral streptococci, staphylococci and lactobacilliit was used in the following experiments.

Effect on bacterial viability

Streptococcus mutans OMZ 175 was suspended in70 mmol/L phosphate buffered saline (pH 7.2) togi ve a concentration of 7.3 1 07 cells/mL. Theethanolic solution of THMC was added to the cellsuspension to give a concentration of 30.0 µg/mL.For the control, only ethanol (1.0 per cent, v/v) wasadded. Changes in the number of viable cells weredetermined at specific time intervals after incubationat 37°C using Mitis-Salivarius agar plates.,

Leakage of cellular substances

The ethanolic solution of THMC was added to S.mutans OMZ 175 (1 108 cells/mL) suspended in70 mmol/L phosphate buffered saline (pH 7.2) togive a concentration of 25.0 µg/mL. The suspensionwas stirred at room temperature, and 1.0 mLaliquots of the cell suspension were filtered througha millipore filter (pore size of 0.22 µm) at specifiedtime intervals. The filtrate was extracted with anequal volume of chloroform, and then theabsorbance of an aqueous phase was measured at260 nm.10

Results

The MIC values of three chalcones for thebacteria are summarized in Table 1. Anti-bacterial

344 Australian Dental Journal 1997;42:5.

hours to obtain THMC. The 2-hydroxychalcone(HC) was available commercially.§

Determination of minimum inhibitoryconcentration (MIC)

All the bacterial strains used (Table 1) were stockcultures from the laboratory at Asahi University.After growing on brain heart infusion (BHI) agar,,they were suspended in fresh BHI broth to give aconcentration of approximately 1 108 cells/mL.

The ethanolic solutions of all chalcones wereadded to BHI agar medium (concentration range25.0-100.0 µg/mL of each). BHI agar was usedthroughout all assays9 because of the poor growth ofstreptococci on Mueller Hinton agar. Control disheswere similarly prepared by adding only ethanol (thefinal ethanolic concentration of 1.0 per cent, v/v),which did not influence the bacterial growth. Theagar plates streaked with the bacterial suspensionswere anaerobically incubated at 37°C for 48 hours.The MIC values were defined as the concentrationsat which no colony was observed after incubation.

§Tokyo Kasei, Tokyo, Japan.,Difco, Detroit, MI, USA.

Fig. 1.–Chemical structures of anti-candidal chalcones.

Table 1. Minimum inhibitory concentrations (MICs) of chalcones against bacteria

Minimum inhibitory concentrations (µg/mL)

Bacteria HC THC THMC

Streptococcus mutans (ATCC 25175) >100.0 100.0 50.0Streptococcus mutans (OMZ 175) >100.0 100.0 50.0Streptococcus mutans (GS 5) >100.0 75.0 50.0Streptococcus sobrinus (ATCC 33478) >100.0 100.0 50.0Streptococcus sobrinus (OMZ 176) >100.0 100.0 50.0Streptococcus cricetus (E 49) >100.0 100.0 50.0Streptococcus salivarius (ATCC 7073) >100.0 50.0 50.0Streptococcus salivarius (ATCC 25975) >100.0 75.0 37.5Streptococcus sanguis (ATCC 10556) >100.0 75.0 37.5Streptococcus sanguis (YST 002) >100.0 75.0 37.5Streptococcus oralis (ATCC 10557) >100.0 75.0 37.5Streptococcus oralis (ATCC 35037) >100.0 100.0 50.0Streptococcus mitis (ATCC 33399) >100.0 75.0 37.5Streptococcus mitis (ATCC 9811) >100.0 50.0 37.5Streptococcus gordonii (ATCC 10558) >100.0 50.0 37.5Lactobacillus casei (ATCC 7469) >100.0 50.0 75.0Staphylococcus aureus (ATCC 25923) 75.0 37.5 <25.01Staphylococcus aureus (ATCC 29213) 100.0 37.5 50.0

Klebsiella pneumoniae (ATCC 15574) >100.0 >100.01 >100.011Klebsiella pneumoniae (G 23238) >100.0 >100.01 >100.011Serratia marcescens (IAM 1161) >100.0 >100.01 >100.011Serratia marcescens (IAM 1240) >100.0 >100.01 >100.011Escherichia coli (ATCC 15489) >100.0 >100.01 >100.011Escherichia coli (US 1) >100.0 >100.01 >100.011

HC: 2-hydroxychalcone, THC: 2,4,2’-trihydroxychalcone, THMC: 2,4,2’-trihydroxy-5’-methylchalcone.

2-Hydroxychalcone 2,4,2’-Trihydroxychalcone 2,4,2’-Trihydroxy-5’-methylchalcone

activity to Gram-positive species was the highest forTHMC, followed by THC and HC. THMC inhibitedthe growth of all streptococci and staphylococci atc o n c e n t r ations lower than 50.0 µg/mL, andLactobacillus casei at 75.0 µg/mL. However, it failedto inhibit the growth of enteric species: Klebsiellapneumoniae, Escherichia coli and Serratia marcescens at100.0 µg/mL. THC inhibited the gr owth ofstaphylococci and L. casei at 37.5 and 50.0 µg/mLr e s p e c t i ve l y, although it required a two - f o l dconcentration over THMC to inhibit most strains ofstreptococci. HC inhibited the growth of only twostrains of staphylococci at 75.0-100.0 µg/mL. Evenat 100.0 µg/mL, it showed no anti-bacterial activityagainst the other species.

The viability of S. mutans was influenced byincubation with THMC at a sub-MIC value ass h own in Fig. 2. Compared with the control,THMC decreased the number of viable cells to1/100 (1 106 cells/mL) in the first 30 minutes.Thereafter, the viability was gradually reduced withi n c u b ation time and the cell number became1/10,000 (6 103 cells/mL) after incubation for threehours.

When S. mutans was incubated with THMC at asub-MIC value, significant amounts of 260 nm-absorbing substances leaked from the cells in thefirst five minutes as shown in Fig. 3. Such a leakagecontinued through subsequent incubations.

Discussion

Microbial adhesion to va rious surfaces is anessential step for the development of infectiousd i s e a s e s. In addition to the direct adhesion todenture base acrylics, plastics and epithelial cells,11-13

C. albicans coaggregates with certain oral bacteriaand this plays an important role in the initial attach-ment of the fungi.8 Increasing evidence has indicatedthat oral streptococci enhance fungal colonizationon denture surfaces. The adhesion of C. albicans toa c rylic strips was confined to areas previouslycovered with Streptococcus sanguis and Streptococcussalivarius.14 By incubating with S. mutans in thepresence of sucrose, larger numbers of C. albicansfirmly adhered to acrylics, which resulted from theglucan-mediated coaggregation between both cells.15

Candida albicans also coaggregated with Streptococcusgordonii, Streptococcus oralis and Streptococcus mitiswhich are the predominant inhabitants of dentureplaque and oral mucosa.16,17 Serum antibody titresnot only for C. albicans but also for S. mutans, S.sanguis and S. mitis were reduced by the treatment ofdenture stomatitis.18,19 Thus, it can be assumed thatoral streptococci participate in the pathogenesis ofdenture stomatitis.

Anti-candidal phytochemicals reported previouslyhave been equivocal in their anti-bacterial activity.3-5

A n t i - b a c t e rial phytochemicals are not so activeagainst C. albicans.5,9 However, THMC inhibitedthe growth of all tested oral streptococci at 50.0-75.0 µg/mL, which corresponded to the MIC valuesfor different strains of C. albicans.7 Both the anti-candidal and anti-streptococcal activities wo u l dmake the phytotherapeutic chalcones, especiallyTHMC, useful in the treatment and prophylaxis ofdenture stomatitis.

The incubation of S. mutans with THMC of asub-MIC value drastically decreased the viable cellcount and almost immediately leaked 260 nm-absorbing substances from the cells. The measure-ment of 260 nm-absorbing substances in supern at a n t

Australian Dental Journal 1997;42:5. 345

Fig. 2.–Effect of 0 (O) and 30 µg/mL (U) 2,4,2’-trihydroxy-5’-methylchalcone (THMC) on the viability of Streptococcus mutans(OMZ 175). Each point indicates the mean SD (n=3-5). Thedifference between control and added THMC was statistically

significant (p<0.001) at all incubation times.

Fig. 3.–Effect of 0 (O) and 25 µg/mL (U) 2,4,2’-trihydroxy-5’-methylchalcone (THMC) on the leakage of 260 nm-absorbingsubstances from Streptococcus mutans (OMZ 175). Each pointindicates the mean SD (n=3-5). The difference between controland added THMC was statistically significant (p<0.001) at all

was frequently used for the determ i n ation ofmaterials such as nucleotide leaking from the cells.10

THMC is likely to show a bactericidal action bychanging the permeability of cellular membranesand damaging their functions, as well as a fungicidalaction.6

Candida albicans synergistically interacts with oralbacteria other than streptococci. In mixed infectionswith S t a p hylococcus aureus, the staphy l o c o c c a lgrowth promoted by C. albicans resulted in elevationof the mortality and morbidity of mice.20 Candidaalbicans has been reported to support the growth ofL. casei in vitro, which in turn restrained candidap r o l i f e r at i o n by the production of lactic acid.21 Inaddition, the fungal enzymatic activity was potentiallyenhanced under such conditions.2 2 The anti-b a c t e rial activity against staphylococci andlactobacilli would be another advantage of THMC.

The chalcones showed no anti-bacterial activityagainst enteric rods at least at the tested concentra-tions. Although piliated enteric rods interact with C.albicans and oral epithelial cells to form a bridgebetween them,23 such species are rarely found in theoral cavity of immunologically normal subjects.2

Antimicrobial agents used for oral infectiousdiseases potentially disturb the normal oral micro-flora which allows the colonization of exogenouspathogens in the oral cavity.2 THMC inhibited thegrowth of C. albicans and oral bacteria at almostcomparable concentrations. Therefore, its topicaluse should not lead to the excess suppression of oralflora.

References01. MacEntee MI. The prevalence of edentulism and diseases

r e l ated to dentures – A literature review. J Oral Rehabil1985;12:195-207.

02. Marsh P, Martin M. Oral microbiology. 3rd edn. London:Chapman & Hall, 1992.

03. Kurosaki F, Nishi A. Isolation and antimicrobial activity of thep hytoalexin 6-methoxymellein from cultured carrot cells.Phytochemistry 1983;22:669-72.

04. Ghannoum MA. Studies on the anticandidal mode of action ofAllium sativum (garlic). J Gen Microbiol 1988;134:2917-24.

05. Heisey RM, Gorham BK. Antimicrobial effects of plant extractson Streptococcus mutans, Candida albicans, Trichophyton rubrumand other microorganisms. Lett Appl Microbiol 1992;14:136-9.

06. Sato M, Tsuchiya H, Akagiri M, et al. Growth inhibitoryp r o p e rties of chalcones to candida. Lett Appl Microbiol1994;18:53-5.

07. Tsuchiya H, Sato M, Akagiri M, Takagi N, Tanaka T, Iinuma M.Anti-candida activity of synthetic hydroxychalcones. Pharmazie1994;49:756-8.

08. Hamada T, Nikawa H. Denture plaque. Tokyo: Ishiyaku, 1991.

09. Tsuchiya H, Sato M, Iinuma M, et al. Inhibition of the growthof cariogenic bacteria in vitro by plant flavanones. Experientia1994;50:846-9.

10. Amin M, Kurosaki F, Nishi A. Carrot phytoalexin alters themembrane permeability of Candida albicans and multilamellarliposomes. J Gen Microbiol 1988;134:241-6.

11. King RD, Lee JC, Morris AL. Adherence of Candida albicansand other Candida species to mucosal epithelial cells. InfectImmun 1980;27:667-74.

12. M i n a gi S, Miyake Y, Inagaki K, Tsuru H, Suginaka H.Hydrophobic interaction in Candida albicans and Candida tropi -calis adherence to various denture base resin materials. InfectImmun 1985;47:11-4.

13. Rotrosen D, Calderone RA, Edward Jr JE. Adherence ofCandida species to host tissues and plastic surfaces. Rev InfectDis 1986;8:73-85.

14. Verran J, Motteram KL. The effect of adherent oral streptococcion the subsequent adherence of Candida albicans to acrylic invitro. J Dent 1987;15:73-6.

15. Branting C, Sund ML, Linder LE. The influence of Streptococcusmutans on adhesion of Candida albicans to acrylic surfaces invitro. Arch Oral Biol 1989;34:347-53.

16. Bagg J, Silve r wood RW. Coagglutination reaction betwe e nCandida albicans and oral bacteria. J Med Microbiol1986;22:165-9.

17. Jenkinson HF, Lala HC, Shepherd MG. Coaggregation ofStreptococcus sanguis and other streptococci with C a n d i d aalbicans. Infect Immun 1990;58:1429-36.

18. Fouche MH, Slabbert JCG, Coogan MM. Candidal antibodiesin patients undergoing treatment for denture stomat i t i s. JProsthet Dent 1987;57:587-91.

19. Fouche MH, Slabbert JCG, Coogan MM. Bacterial antibodiesin patients undergoing treatment for denture stomat i t i s. JProsthet Dent 1987;58:63-8.

20. Carlson E. Synergistic effect of Candida albicans a n dS t a p hylococcus aureus on mouse mort a l i t y. Infect Immun1982;38:921-4.

21. Young G, Krasner RI, Yudofsky PL. Interactions of oral strainsof Candida albicans and lactobacilli. J Bacteriol 1956;72:525-9.

22. S a m a r a n ayake LP, Raeside JM, MacFarlane TW. Fa c t o rsaffecting the phospholipase activity of Candida species in vitro.Sabouraudia 1984;22:201-7.

23. Centeno A, Davis CP, Cohen MS, Warren MM. Modulation ofCandida albicans attachment to human epithelial cells by bacteriaand carbohydrates. Infect Immun 1983;39:1354-60.

Address for correspondence/reprints:Department of Oral Microbiology,

Asahi University School of Dentistry,1851-1 Hozumi, Hozumi-cho,

Motosu-gun,Gifu 501-02,

Japan.

346 Australian Dental Journal 1997;42:5.