Antimicrobial Activity of Cratoxylum Formosum on Streptococcus Mutans
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SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH ANTIMICROBIAL ACTIVITY OF CRATOXYLUM FORMOSUM ON STREPTOCOCCUS MUTANS Theeralaksna Suddhasthira1, Sroisiri Thaweboon1, Nartruedee Dendoung2, Boonyanit Thaweboon1 and Surachai Dechkunakorn1 1Faculty of Dentistry, 2Faculty of Social Sciences and Humanities, Mahidol University, Bangkok, Thailand Abstract. The gum of Cratoxylum formosum, commonly known as mempat, is a natural agent that has been used extensively for caries prevention by hill tribe people residing in Thailand. The objective of this study was to investigate the antimicrobial activity of Cratoxylum formosum gum on Streptococcus mutans (S. mutans) in vitro. The gum extracted from stem bark of Cratoxylum formosum was investigated for antimicrobial activity against different strains of S. mutans, including S. mutans KPSK2 and 2 clinical isolates. Inhibition of growth was primarily tested by agar diffusion method. A two-fold broth dilution method was then used to determine the minimum inhibitory concentration (MIC) of the extract. The extract of Cratoxylum formosum was effective against S. mutans with the inhibition zones ranging from 9.5 to 11.5 mm and MIC values between 48 µg/ml and 97 µg/ml. The gum of Cratoxylum formosum has high antimicrobial activity against S. mutans and may become a promising herbal varnish against caries. INTRODUCTION Species of this genus have been used for their diuretic, gastric and tonic effects, as well as Natural products have been used for for diarrhea, flatulence, food poisoning and thousands of years as folk-medicine, and are internal bleeding (Grosvenor et al, 1995). C. promising sources for novel therapeutic agents formosum is widely distributed in northeast- (Cragg et al, 1997). They have been used or ern Thailand. A decoction of the bark is used investigated as promising agents to prevent to cure colic. The gum has been used exten- oral diseases, especially plaque-related dis- sively by hill tribe people of Thailand for paint- eases, such as dental caries (Koo et al, 2002; ing on tooth surfaces as varnish to prevent Hwang et al, 2003). Among various medicinal dental caries. plants used in folk-medicine in Thailand, Dental caries are one of the most com- Cratoxylum formosum, a shrub 10-20 meters mon infectious diseases in the Thai popula- tall known as Tew, stands out because of its tion. The prevalence of dental caries in Thai multiple pharmacological properties. children age 5-6 years in 2000 has been re- Cratoxylum is a small group belonging to ported to be 87.4% with a dmft (decayed the Guttiferae family, distributed in several missing and filled teeth) value of 5.97 (Minis- Southeast Asian countries (Iinuma et al, 1996). try of Public Health, 2002). Streptococcus mutans is closely associated with the patho- Correspondence: Sroisiri Thaweboon, Department genesis of dental caries because of its ability of Microbiology, Faculty of Dentistry, Mahidol Uni- to synthesize water-insoluble glucans that versity, 6 Yothi Road, Bangkok 10400, Thailand. mediate adhesion to and colonization of the Tel: 66 (0) 2644-8644-6 ext 6406-8; Fax: 66 (0) 2203-6405 teeth (Freedman et al, 1978). S. mutans also E-mail: [email protected] produces large amounts of acid, particularly 1156 Vol 37 No. 6 November 2006 ANTIMICROBIAL ACTIVITY OF C. FORMOSUM ON S. MUTANS lactic acid, which are potent in driving tooth the starting inoculum of 1 x 108 CFU/ml. The demineralization (Johnson et al, 1980). There- vehicle (50% DMSO, v/v) was used as a nega- fore, the use of antimicrobial agents to con- tive control and chlorhexidine gluconate was trol these cariogenic bacteria is one of the used as a positive control for growth. The strategies for caries prevention and treatment. final concentration of DMSO was 0.24% (v/v). The objective of this study was to investigate The MIC was defined as the lowest concen- the antimicrobial activity of C. formosum gum tration of extract that had restricted growth to on S. mutans in vitro. a level lower than 0.05 at 600 nm (no visible growth). Each experiment was repeated four MATERIALS AND METHODS times. Plant material and extraction RESULTS The trunk parts of Cratoxylum formosum were collected from mountainous areas of The antimicrobial activity of Cratoxylum northern Thailand, in January-Febuary, 2006 formosum gum extract against 3 strains of S. and authenticated by the herbarium staff of mutans was quantitatively assessed by the the Faculty of Agriculture, Kasetsart Univer- presence of inhibition zone diameters and the sity, Thailand. They were cut and burned. MIC values (Tables 1 and 2). Black gum was collected immediately and transferred directly to a sterilized Petri dish. Microorganisms and antimicrobial activity assay Table 1 The microorganisms used in this study Antimicrobial activity of Cratoxylum formosum gum extract against S. mutans were S. mutans KPSK2, and two recent clinical isolates. The antimicrobial activity was deter- strains tested based on the disk diffusion mined by disk diffusion method and then fur- method. ther investigated for the minimum inhibitory Inhibition zone concentration (MIC). In the disk diffusion sus- S. mutans strain diameter (mm) ceptibility method, Muller-Hinton agar was used Extract Chlorhexidine and prepared according to the instructions of the manufacturer (Oxford, UK). All agar plates KPSK2 11.5 ± 0.4 20.5 ± 0.5 were prepared in 90 mm Petri dishes with Clinical isolate I 11.0 ± 0.9 19.3 ± 0.6 20 ml of agar, giving a final depth of 4 mm. Clinical isolate II 9.5 ± 0.4 19.5 ± 0.5 Overnight broth cultures were prepared in Brain heart infusion broth (Difco), buffered Table 2 with PBS (phosphate buffer saline, pH 7.4) in The MIC value of Cratoxylum formosum order to yield approximately 1.5 x 108 CFU/ gum extract against S. mutans strains on ml. Whatman paper disks (Whatman Interna- the tube dilution assay. tional, UK) 6 mm diameter were placed on the inoculated agar surfaces and were impreg- S. mutans strain MIC (µg/ml) nated with 20 µl of the extract. For MIC deter- Extract in Chlorhexidine mination, the gum extract of C. formosum was 50%DMSO dissolved in DMSO (dimethylsulfoxide, 50% v/v) in order to overcome the limited solubility KPSK2 48 <1.15 of the gum in aqueous media. Two-fold dilu- Clinical isolate I 97 <1.15 tion series of the extract were tested against Clinical isolate II 97 <1.15 Vol 37 No. 6 November 2006 1157 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH DISCUSSION antimicrobial activity against both gram-posi- tive and gram-negative bacteria, such as Ba- Applying a black varnish to the teeth has cillus subtilis, Staphylococcus aureus, been observed as a habit in hill tribe people Pseudomonas aeruginosa, Streptococcus residing in northern Thailand. This habit is a faecalis and Samonella typhi while anthraquino- wide-spread Asian tradition, not only in Thai- nes have little activity. Not much is known about land but also in Lao PDR, Vietnam, Myanmar, the antimicrobial activity of C. formosum against Cambodia and China (Nguyen, 1990). It has oral microorganisms. Tayanin and Bratthall been maintained for many countries, recog- (2006) found little inhibitory effect of C. nized as a symbol of elegance and good taste. formosum on the growth of salivary Mutans The techniques and materials used vary by streptococci (MS). In their study, plastic strips country and population. of the Strip Mutans Test kit were partly cov- Cratoxylum formosum or Tew is a Thai ered with the extract and stimulated saliva from medicinal plant used in folk-medicine as an persons with high MS levels was added. After agent to prevent oral diseases, especially den- incubation, a few colonies were observed. It tal caries. Hill tribe people use black gum from should be noted that the strip method used in the burned stem bark to stain their teeth by their study is not the standard method to in- smearing it on the buccal and occlusal sur- vestigate antimicrobial activity. In addition, the faces. It is believed to have dental preserva- amount of extract and the concentration of MS tive effect against decay and pain. in saliva were not indicated in their study. In this investigation, gum extracted from Natural products have recently been stud- C. formosum showed high antimicrobial ac- ied as an alternative to synthetic chemical sub- tivity against S. mutans with an MIC value be- stances for dental caries prevention. Black tween 48 µg/ml and 97 µg/ml. According to gum from the burned bark of C. formosum is Rios et al (1988), natural crude extracts that considered as a promising candidate. Further exhibit activity at concentrations lower than studies are needed to clarify the bioactive 100 µg/l may have great antimicrobial poten- compound in this plant extract. tial, since the active compounds can be iso- In conclusion, the remarkable inhibitory lated and used at lower concentrations. The effects of C. formosum gum extract suggest MIC values for the clinical isolates were higher that this plant may be a useful source for the than those of the culture collection strain. This development of a novel herbal varnish against observation is important because the labora- dental caries. Further studies should be con- tory strains commonly used to determine sus- ducted to examine whether this extract has ceptibility to antimicrobials, may not express any influence on the viability of S. mutans or the same virulence or resistance patterns the development of dental biofilms. compared to the strains recently isolated from the oral cavity (Duarte et al, 2003). It is note- ACKNOWLEDGEMENTS worthy that the extract was effective against recently isolated bacteria. The authors wish to express their sincere The antimicrobial activity of an agent may thanks to Assoc Prof Surin Soo-Ampon for his be indicative of the presence of metabolic tox- valuable advice and encouragement.