Tooth decay is a prevalent dental disease and Streptococcus mutans and Lactobacillus rhamnosis are the most important bacterial causes of tooth decay. Because of the increased antibiotic resistance of bacteria and the side effects of antibiotics, this study was conducted to investigate the antibacterial effect of hydroalcoholic Teucrium Chamaedrys extraction S.mutans and L.rhamnosis. In this experimental-laboratory study, T.chamaedrys was extracted by maceration, and the standard strains of lyophilized S.mutans and L.rhamnosis were provided from Iranian Research Organization for Science and Technology for investigation of T.chamaedrys effect on their growth. The antibacterial effect of hydroalcoholic T.chamaedrys was investigated on S.mutans and L.rhamnosis and then minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The MIC of T. chamaedrys was obtained 128 and 32 μg/L against S.mutans and L.rhamnosis, respectively. The MBC of T.chamaedrys was obtained 256 and 64 μg/L against S.mutans and L.rhamnosis, respectively. T.chamaedrys bactericidal and bacteriostatic effects on L.rhamnosis and S.mutans, two bacterial causative agents of dental cavity show that it might be used for prevention of dental cavity due to these patogens

Derakhshan, Ali.

Gholipour, Abolfazl.

Heidari Soureshjani, Reza.

Khaledi, Mansoor.

shahrekord university of medical scinces

ISSN: 0974-2115 www.jchps.com                                                                    Journal of Chemical and Pharmaceutical Sciences October - December 2016 3419 JCPS Volume 9 Issue 4 Ibactericidal and Bacteriostatic In Vitro Effects of Teucrium Chamaedrys Hydroalcoholic Extract on Two Bacterial Causative Agents of Tooth Decay Reza Heidari Soureshjani1, Ali Derakhshan2*, Mansour Khaledi 1, Abolfazl Gholipour 3 1Students Research Committee, Shahrekord University of Medical Sciences, Shahrekord, Iran 2Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran 3Department of Microbiology and Immunology and Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran. *Corresponding author: E-Mail: dr.aliderakhshan@yahoo.com ABSTRACT Tooth decay is a prevalent dental disease and Streptococcus mutans and Lactobacillus rhamnosis are the most important bacterial causes of tooth decay. Because of the increased antibiotic resistance of bacteria and the side effects of antibiotics, this study was conducted to investigate the antibacterial effect of hydroalcoholic Teucrium Chamaedrys extraction S.mutans and L.rhamnosis. In this experimental-laboratory study, T.chamaedrys was extracted by maceration, and the standard strains of lyophilized S.mutans and L.rhamnosis were provided from Iranian Research Organization for Science and Technology for investigation of T.chamaedrys effect on their growth. The antibacterial effect of hydroalcoholic T.chamaedrys was investigated on S.mutans and L.rhamnosis and then minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The MIC of T. chamaedrys was obtained 128 and 32 µg/L against S.mutans and L.rhamnosis, respectively. The MBC of T.chamaedrys was obtained 256 and 64 µg/L against S.mutans and L.rhamnosis, respectively. T.chamaedrys bactericidal and bacteriostatic effects on L.rhamnosis and S.mutans, two bacterial causative agents of dental cavity show that it might be used for prevention of dental cavity due to these patogens.  KEY WORDS: Hydroalcoholic extract, Teucrium chamaedrys, Streptococcus mutans, Lactobacillus rhamnosis 1. INTRODUCTION Tooth decay is a prevalent infectious disease in children (Olak, 2007). Studies have reported that different factors are the risk factors for tooth decay, including oral micro flora and health habits (Gudkina and Brinkmane, 2010).Tooth decay which causes demineralization of oral tissues is a dental bacterial infection (Gudkina and Brinkmane, 2010). Nowadays, certain streptococcal species, such as Streptococcus mutans, and some lactobacillus species, including Lactobacillus rhamnosis, are considered important causative causative agents of dental plaques and caries (Thorild, 2002). L.rhamnosis and S.mutans are gram-positive and obligate anaerobic cocci (Clifford and Stuedevand, 1995). S.mutans is the most important and first microorganism known to cause tooth decay, and is present in plaques. To date, over 25 species of S.mutans have been demonstrated to cause loss of tooth enamel by producing lactic acid and fermenting sucrose. There has been no project to definitely exterminate tooth decay to date, and dental expenses were estimated approximately 70.3 billion dollars in the USA in 2003. There are over 2000 species of Teucrium with confirmed analgesic, anti-inflammatory, anticonvulsant, antipyretic and anti-burn properties, but some of them are toxic (Ansari, 2013, Baradaran, 2013). According to Iranian traditional medicine, Teucrium Chamaedrys is used as a disinfectant, bactericidal, and antipyretic agent. T.chamaedrys is a perennial, herbaceous plant with many branches, reaches to a height of 10 to 35 cm and is white as cotton. It usually occurs in barren areas, rocky shores and sandy regions across the world. Its leaves are narrow and long. The flowers are white, yellowish white, yellow and even purple. Flowering shoot is the usable part of this plant (Najafi F). T.chamaedrys is from Lamiaceae family. There are 12 species of Teucrium in Iran, of which three are exclusive to Iran. Because of the increased antibiotic resistance of bacteria and the side effects of antibiotics, the use of medicinal plants and is important. Medicinal plants are considered as a good source for preparation of new drugs (Delfan, 2014, Baharvand-Ahmadi, 2015, Saki, 2014, Nasri and Shirzad, 2013) . As well, medicinal plants have several benefits such as low tocxicity, low cost, availability, and agreeability to human nature (Sewell and Rafieian-Kopaei, 2014). According to ethnobotanical investigations in Lorestan, Iran, T.chamaedrys can be used to prevent tooth decay. Because S.mutans is a significant bacterial causative agent of tooth decay and the effect of T.chamaedrys has not yet been investigated on S.mutans, this study was conducted to investigate the antibacterial effect of  hydroalcoholic T.chamaedrys extraction S.mutans and L.rhamnosis. 2. MATERIALS AND METHODS First, T.chamaedrys was provided from Larestan, southern Fars province, southern Iran. After identification and scientific confirmation of the plant, the samples were dried and pulverized. Extraction was done by maceration. For this, 100g of the plant was dissolved with one L of alcohol 80%. The solution was kept in darkness for 72 hours. After filtering with Whatman filter paper 0.5 in duplicate, the solution was distilled with rotary evaporator (vacuum distillation) and then incubated at 37°C. Finally, a honey-thick extract was ontained. A stock solution of this extract was prepared with dimethyl sulfoxide (DMSO). ISSN: 0974-2115 www.jchps.com                                                                    Journal of Chemical and Pharmaceutical Sciences October - December 2016 3420 JCPS Volume 9 Issue 4 In this work, the standard strains of lyophilized S.mutans, PTCV35668 and ATCC1683, and lyophilized L.rhamnosis, PTCC1637 and ATCC53103, were provided from the Iranian Research Organization for Science and Technology. To prepare microbial suspension, 1.5×10 8CFU/mL 24-h culture was conducted on blood agar, and then a suspension with 0.5 Mc Farland opacity was prepared in normal saline. The antibacterial effect of hydroalcoholic T.chamaedrys extract was investigated on S.mutans and L.rhamnosis according to Broth Microdilution technique. Minimum inhibitory concentration (MIC) was tested in 96-well, sterile plates according to two-fold Broth Microdilution technique and the Clinical & Laboratory Standards Institute in triplicate.   First, a stock solution of the extract was prepared. In this work, the serial, two-fold dilutions, from 1 to 500 µg/mL, were used. The determined dilutions were conducted in 96-well plates in triplicate for each sample. Then, 95 µL of Mueller-Hinton agar was introduced into each well. Afterwards, 5 µL of bacterial suspension, equal to .0 5 Mc Farland, and then 100 µL of serial two-fold dilutions of hydroalcoholic T.chamaedrys extractwas introduced into all the wells. The final volume of each well was 200 µL. After mixing the samples with a shaker at 300 rpm for 20 seconds, we incubated them at 37°C for 18-24 h. Then, optical densities of the samples were read at 450 nm wavelength with an ELISA reader (State Fax 2100, USA). No development of opacity was considered representative of minimum inhibitory concentration (MIC). Minimum bactericidal concentration (MBC) of the ethanolic T.chamaedrys extract was determined by Broth Micro dilution. To determine the MBC of each extract, nine sterile test tubes were used. Of these test tubes, eight were used to test different dilutions of each extract and one was considered control. After culture, all of the test tubes were incubated at 37°C for 24 h. After incubation, the tubes were investigated for the opacity due to inoculated microorganism’s growth, specimens were taken from all the tubes in which no growth was observed, and cultured for determination of the MBC. The tubes with the lowest concentrations of the extract and no growth in the corresponding plates were considered MBC. The well containing 195 µL of the extract-free culture medium and five µL of bacterial suspension was considered negative control. Different concentrations of amikacin besides Mueller-Hinton Broth and five µL of bacterial suspension were considered positive control.  3. RESULTS Determination of the MIC and MBC of hydroalcoholic T.chamaedrys extract against S.mutans and L.rhamnosis by Broth Microdilution technique, indicated that two, four, eight, 32, and 64 µg/L of S.mutans were resistant to T.chamaedrys, but 128 µg/L and higher concentrations of S.mutans were susceptible to T.chamaedrys. Two, four, and eight µg/L of L.rhamnosis were resistant to T.chamaedrys, but 32 µg/L and higher concentrations of L.rhamnosis were susceptible to T.chamaedrys (Table 1). Table.1. MIC of T.chamaedrys on S.mutans and L.hamnosis T.chamaedrys concentration (mg/mL) Microorganism Type of extract Control 256 128 64 32 8 4 2  S.mutans  Ethanolic - + + - - - - - - + + + + - - - L.rhamnosi Ethanolic Therefore, the most desirable MIC was obtained 128 and 32 µg/L against S.mutans and L.rhamnosis, respectively. The most desirable MBC was obtained 128 and 64 µg/L against S.mutans and L.rhamnosis, respectively (Table 2). Table.2. MBC of T.chamaedrys on S.mutans and L.rhamnosis T.chamaedrys concentration (mg/mL)  Microorganism Type of extract Control 256 128 64 32 8 4 2  S.mutans  Ethanolic - + - - - - - - - + + + - - - - L.rhamnosi Ethanolic  DISCUSSION As previously stated, S.mutans and L.rhamnosis are the most important causative agents of tooth decay. Tooth decay is indeed an infectious-microbial disease that causes demineralization of calcareous oral tissues. Tooth decay occurs in a specific site of tooth when the population of S.mutans bacteria reaches to 50% of all bacterial population in that site. Therefore, changes in oral microbial ecology are considered important to prevent tooth decay by decreasing S. mutans and L.rhamnosis populations.  Acidogenic bacteria, such as S.mutans, increase tooth decay dramatically  (Fabricant and Farnsworth, 2001). In the present study, hydroalcoholic T.chamaedrys extract exerted limited in vitro antibacterial effects on S.mutans. ISSN: 0974-2115 www.jchps.com                                                                    Journal of Chemical and Pharmaceutical Sciences October - December 2016 3421 JCPS Volume 9 Issue 4 In contrast, in a study on methanol and oil extracts of Ziziphora clinopodioides, the methanol Z. clinopodioides extract exerted inhibitory effects on the growth of a number of gram-positive and negative bacteria (Chitsaz, 2006). A study on in vitro effects of polyphenols in some plants, including tea, demonstrated that these effects were dose-dependent on some bacterial species (Taguri, 2004). However, in the present work, the MIC of T.chamaedrys was determined higher than 128 and 32 µg/mL against S.mutans and L.rhamnosis, respectively.   Investigations on antibacterial and antifungal properties of hydroalcoholic extract of Salvia officinalis leaves, Mentha pulegium, and Pimpinella anisum demonstrated that these plants exerted stronger antibacterial effects than T.chamaedrys on bacterial causative agents of tooth decay, as the MICs of S. officinalis, M. pulegium, and P. anisum were derived 6.25, 12.5, and 12.5 µg/mL against S.mutans and 1.56, 3.12, and 12.56 µg/mL against L.rhamnosis, respectively (Kermanshah H). Moreover, a study on green and black tea extracts demonstrated that both types of teas had bactericidal property, but bactericidal property of black tea was greater on oral Streptococci than green tea (Hamdi, 2008). In contrast, the present work demonstrated that hydroalcoholic T.chamaedrys extract exerted a smaller inhibitory effect on the bacterial causative agents of tooth decay compared to the plants investigated in that study (Hamdi, 2008). Regarding the findings of the present study and other works, we can argue that T.chamaedrys hydroalcoholic extracts has antibacterial effects on S.mutans and L.rhamnosis and might be beneficial for prevention of tooth decay. The antimicrobial mechanism of these plants is not clear. Phenolic compounds which are abundant in this plant have antimicrobial activity (Sharafati-Chaleshtori and Rafieian-Kopaei, 2014, Bahmani, 2014, Chaleshtori, 2011, Karamati, 2014, Rahimian, 2013) and these compounds might be responsible for the plant effect. There are numerous plants which possess phenolic compounds (Shanafelt, 2002; Sarrafchi, 2016; Bahmani, 2016; Shayganni, 2015; Rahimi-Madiseh, 2016; Taherikalani, 2016). These plants also might have antimicrobial activities which should be examined.    4. CONCLUSION Antibacterial effects of hydroalcoholic T.chamaedrys extract on S.mutans and L.rhamnosis causative agents of tooth decay is limited. Hence, regarding the limited antibacterial effect of hydroalcoholic  T.chamaedrys extract on S.mutans and L.rhamnosis, and consequently tooth decay, we recommend that other bactericidal plants be investigated in future works to offer an economical treatment, with minimal side effects, for tooth decay. Moreover, future studies are recommended to investigate effects of simultaneous use of this plant and different concentrations of other bactericidal plants, and compare these plants' effects and antibiotics'.  5. ACKNOWLEDGMENTS This article was obtained from a research project approved and registered at the Research and Technology Deputy of the Shahrekord University of Medical Sciences (no. 1496). Hereby, we gratefully thank all people who helped us to conduct this study.  REFERENCES Ansari R, Shahinfard N, Namjou A, Rafieian M, Shirzad H, Rafieian-kopaei M. Ameliorative property of Teucrium polium on second degree burn. J Herbmed Plarmacol, 2(1), 2013, 9-11. 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Ibactericidal and bacteriostatic in vitro effects of teucrium chamaedrys hydroalcoholic extract on two bacterial causative agents of tooth decay

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shahrekord university of medical scinces