Effect of P.betle in Dentifrice on Streptococcus mutans of Schoolchildren Sutadi H*.,Desiree S*.,Anni.TD*.,Felicia S*., Mangundjaja S** *Department of Paediatric Dentistry and **Department of Oral Biology Faculty of Dentistry Universitas Indonesia Jakarta Indonesia Abstract Piper betle has active substance : Atsiri oil known as antiseptic agent which is beneficial for oral health. One of its benefits is capability to inhibit the growth of Streptococcus mutans Objective: The aim of the study is to determine the sensitivity of Piper betle (P.betle) in dentifrice on mutans streptococci Methods: The effect of Piper betle in dentifrice was examined in vitro on the inhibit the bacterial growth by determining the inhibition zone (agar diffusion method), minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC).The microorganisms tested were composed 6 strains of Streptococcus mutans isolated from schoolchildren in Jakarta Indonesia.. Data obtained was done in a descriptive method. Results: showed that Piper betle in dentifrice had effect on all of mutans of Streptococcus mutans1(inhibition zone 4.50mm; MIC 10-3./ml,MBC10-1./ml); Streptococcus mutans2 (inhibition zone 3.00mm; MIC 10-3./ml,MBC10-1./ml); Streptococcus mutans3(inhibition zone 2.10mm; MIC 10-3./ml,MBC10-1./ml); Streptococcus mutans4(inhibition zone 2.90mm; MIC 10-3 /ml,mbc10-1./ml); Streptococcus mutans5(inhibition zone 2.20mm; MIC 10-3./ml,MBC10-1/ml), Streptococcus mutans6(inhibition zone 2.80 mm; MIC 10-3./ml,MBC 10-1./ml) and their MBC value was three times bigger than MIC. Conclusion: We concluded that Piper betle in dentifrice has anti microbial activity against local strains of Streptococcus mutans isolated from schoolchildren. Hence, it may have potential anti-caries properties. Key words: Piper betle - dentifrice - Streptococcus mutans Introduction Piper betle Linn leaves have long been used in Indonesia as traditional medicine. These leaves have antimicrobial activity towards bacteria in the mouth i.e. Streptococcus viridans, Staphylococcus aureus and Streptococcus mutans. Dental caries,which is known the most common ailment of the mouth, is found to be caused by Mutans Streptococci. The presence of Streptococcus mutans is associated with the high number of salivary mutans streptococci 2 And caries activity of mutans streptococci of mutans streptococci in high caries prevalence 3 Various effort to prevent dental caries have been done. The simplest and common effort is tooth brushing with toothpaste or dentifrice. Recently, many kinds of dentifrice have been found in the market with many kinds of brands and
utilities, so consumers are demanded to select one of that most beneficial for oral cavity hygiene. The objective of the study is determining the sensitivity of toothpaste containing Salvadora persica by measuring the inhibitory zone, minimum inhibitory concentration (MIC) and minimum bactericidal concentration(mbc) on mutans streptococci, in vitro. This study is expected to contribute significantly to world of science that toothpaste containing Salvadora persica can inhibit the growth of mutans streptococci in plaque, therefore in long term of using dentifrice containing Salvadora persica, caries can be prevented. Materials and Methods The material used in this study is tooth paste containing Salvadora persica was examined in vitro. The microorganisms tested were local strains of mutans of Streptococcus mutans isolated from humans composed 6 strains of Streptococcus mutans isolated from humans in Bangka Island Indonesia. In this case, those mutans streptococci are cultivated in TYS20B 4, BHI (Brain Heart Infusion Broth) and diagnostic sensitivity test (DST) was performed.those specimen are incubated in anaerobic jar at 37 0 Celsius for 3 X 24 hours. Working method Sensitivity test to antibiotic can be done in two ways 5 : I. Drug serial dilution method : a. Making the bacteria culture medium a.1. From the cultivated mutans streptococci in TYS20B, take one loop of bacteria and cultivate them in liquid culture medium BHI, then incubated it in anaerobic jar at 37 0 Celsius degree for 2 X 24 hours. a.2. After 2 days, compare the turbidity of bacteria media culture of BHI with Brown III standard solution. a.3 If it is found that bacteria culture in BHI media is more turbid, add sterile saline solution, little by little until the turbidity is equal to Brown III standard solution. a.4. If the turbidity of bacteria culture medium is equal to Brown III standard solution, the number of bacteria cell/ml in bacteria culture medium can be counted, their number is + 9 X 10 8 bacteria /ml. b. Bacteria dilution If equalization process has been done, culture of mutans streptococci bacteria shall be diluted as follows: b.1. Prepare 7 tubes of each 9 ml containing physiologic saline solution, and also prepare 1 tube containing 5 ml physiologic saline solution. 2
b.2. Take 1 ml bacteria from item a.5, the put into the first tube, shake them thoroughly, from the first tube take 1ml bacteria culture and put into the second tube, and do the same thing through the seventh tube. b.3 Take 5 ml of bacteria from the seventh tube and put them into the eight tube and shake it thoroughly. b.4. The number of bacteria is estimated + 50 cells/ml each tube shall be labeled. c. Sensitivity test of bacteria to Salvadora persica in Salvadora persica dentifrice. c.1. Prepare 5 sets of test tubes, each tube is filled with 9 ml BHI and is labeled 1 5 respectively. c.2. Put 1 gram of Salvadora persica s tooth paste as much with 1 : 1 concentration into first tube then stir it well. c.3. From the first tube, take 1 ml of solution and put into the second tube, do the same thing through the fifth tube. c.4. After dilution finished, then put 1 ml of diluted Streptococcus mutans from item b.4 in those five test tube. All test tubes put in anaerobic jar at 37 0 Celsius degree for 2 X 24 hours. C.5. After 72 hours macroscopically we can see in which tube, the bacteria can not growth. Record the result to determine the Minimum Inhibition Concentration (MIC) II. Method using disk with drug in solid media II.1 Diluted 1 ml of mutans streptococci in the agar DST petri disc, the bacteria suspension wet the DST agar thoroughly. II.2. Then put dilution of Salvadora persica toothpaste on a disk and put it on the surface of DST agar. II.3. Those petri discs are incubated in anaerobic jar at 37 0 Celsius degree for 3 X 24 hours. II.3. Inhibition zone will show around the disk and measuring the diameter of the isolated zone around the samples. Data obtained is analyzed in a descriptive. Results of Research Table 1 showed that The Minimum Inhibitory Concentration MIC is 10-3 /ml and MBC 10-1 /ml for Streptococcus mutans1, Streptococcus mutans2, Streptococcus mutans3, Streptococcus mutans4, Streptococcus mutans5 and Streptococcus mutans6. 3
Table1. Results of serial dilution test of Streptococcus mutans to Salvadora persica dentifrice The concentration of Salvadora persica toothpaste (/ml) Type of bacteria 01 0.01 0.001 0.0001 0.00001 (I) (II) (III) (IV) (V) C(+) C(-) S.mutans1 - - - + + + - S.mutans2 - - - + + + - S.mutans3 - - - + + + - S.mutans4 - - - + + + - S.mutans5 - - - + + + - S.mutans6 - - - + + + - + growing - not growing C(+)Positive control without Salvadora persica toothpaste) C(-) Negative control (with Salvadora persica) Table 2 The measurement of inhibitory zone which is carried out from the border of disk to zone with bacterial growth showed that Inhibitory zone of Streptococcus mutans1 is 4.00 mm; 2.50 mm for Streptococcus mutans2; 1.90 mm for Streptococcus mutans3; 2.00 mm for Streptococcus mutans4; 1.50 mm for Streptococcus mutans5,1.50 mm for Streptococcus mutans6 Table 2. The result on inhibitory zone measurement in bacterial growth of mutans streptococci on DST agar media. Type of Bacteria Streptococcus.mutans1 Streptococcus.mutans2 Streptococcus. mutans3 Streptococcus.mutans4 Streptococcus.mutans5 Streptococcus.mutans6 Inhibitory zone in mm 4.00 2.50 1.90 2.00 1.50 1.50 Average of inhibitory zone of local strain Streptococcus mutans : 2.23 mm 4
Discussion From the results showed that all of local strains of Streptococcus mutans1, Streptococcus mutans2,streptococcus mutans3,streptococcus mutans4, Streptococcus mutans5 and Streptococcus mutans6 are sensitive to concentration 10-1 /ml Salvadora persica dentifrice Inhibitory zone of Streptococcus mutans1 is 4.00 mm; 2.50 mm for Streptococcus mutans2; 1.90 mm for Streptococcus mutans3; 2.00 mm for Streptococcus mutans4; 1.50 mm for Streptococcus mutans5, 1.50 mm for Streptococcus mutans6 Currently known that Streptococcus mutans are triggering factor of caries because their characteristics always are related to caries process. Streptococcus mutans can synthesize insoluble glucan (1-3) bond from sucrose 6, besides that Streptococcus mutans are also more aciduric than other Streptococci group 7. Dentifrice containing Salvadora persica that one of its benefits is capability to prevent dental caries because it has active substance: Tritely Amine, Alkaloid, Chlorine, Fluoride, Saponin, Tannin, Resin, Sulfur, Vitamin C and Sterol 1. Due to the condition the use of Salvadora persica dentifrice might inhibit the growth the population of mutans of Streptococcus mutans in plaque and saliva. In this study the author examined antibacterial effect of commercial dentifrice available in the market on mutans of Streptococcus mutans using sensitivity test, in vitro. If dentifrice does posses antibacterial effect on mutans streptococci, it is expected that the use of dentifrice in oral cavity can reduce population of Streptococcus mutans in plaque so that its harmful product can be reduced too. It is hope that caries will decrease significantly. Conclusion The result showed that an Salvadora persica in Salvadora persica dentifrice has bacterial activity against local strains of mutans of Streptococcus mutans isolated from humans harbouring species in Bangka Island Indonesia It is expected that it can be used in preventing caries risk in the future 1. What is Siwak-F. Available at http://www.siwak-f.com. 2005 2. Mangundjaja S., Muthalib A., Djais A Streptococcus mutans in Caries Free and Caries Active Subject in Panggang Island Indonesia. Paper read at The International Association for Dental Research General Assembly 1995 Singapore 3. Mangundjaja S., Muthalib A.Activity Streptococcus mutans Isolated from Human Harboring Species in Kelapa Island Indonesia Paper read at FDI Conference 1995 Hong Kong 4. Schaeken M.J.M., van der Hoeven J.S.,Franken H.C.M. Comparative Recovery of Streptococcus mutans on Five Isolation Media Including A New Simple Selective Medium. J.Dent. Res 1986 ;65: 906-8 5
5. Bailey WR,Scott EG. Diagnostic microbiology, 3 rd ed. St. Louis: CV Mosby.1970 6. Heriandi.S,. The Determination of the Predictive Value of Caries Activity Test Cariostat. 13 th Congress of International Association of Dentistry for Children Kyoto Japan.1992 7..Kohler B.,Birkheid.,Olsson S. Acid production by human strains of Streptococcus mutans and Streptococcus sobrinus Journal Caries Res 1995;29:402-6 ----000--- Presented at The 1th International Pediatric Dentistry Conference 24 25 November 2006 Universiti of Kebangsaan Malaysia Kuala Lumpur. 6