Wesal F.doc J Bagh College Dentistry Vol. 25(4), December 2013 Effect of small cardamom Pedodontics, Orthodontics and Preventive Dentistry160 Effect of small cardamom extracts on Mutans streptococci in comparison to chlorhexidine gluconate and de-ionized water (In vitro study ) Ghada A. Ibrahim, B.D.S. (1) Wesal A. Al – Obaidi, B.D.S., M. Sc. (2) ABSTRACT Background: Herbs are being widely explored to discover alternatives to synthetic antibacterial agents.Small Cardamom often referred to as queen of spices because of its very pleasant aroma and taste, have a history as old as human race. Most people use cardamom as a spice and are largely unaware of its numerous health benefits. The purpose of this study was to evaluate the effect of different concentrations of water and alcoholic cardamom extracts on sensitivities, growth, and adherence of Mutans streptococci in vitro. Materials and Methods: In this study, saliva was collected from ten volunteers (College students 18-22 years). Agar well technique was used to study the sensitivities of Mutans streptococci to different concentrations of small cardamom extracts and other control agents, also the effects of small cardamom extracts on viable counts, adherence of Mutans streptococci were studied Results: According to agar well diffusion methods, both cardamom extracts were effective in inhibition of Mutans streptococci, but still weaker than chlorhexidine gluconate 0.2%. Alcoholic extracts showed higher zone of inhibition compared to the same concentration of water with high significance differences (P<0.01). The effects of 10%, 15%, and 20% of both water and alcohol extracts of small cardamom were tested on the viability counts of Mutans streptococci in vitro. Highly significant reduction in the counts of bacteria was reported of both cardamom extracts and CHX in comparison to control without agents after 24 hr. Both cardamom extracts less effective than CHX. All the concentration of water and alcohol cardamom mouth washes tested was not effective in prevention the adherence of bacteria on teeth surface in vitro, while CHX was effective in prevention the adherence of bacteria. Conclusion: Cardamom extracts were effective against Mutans streptococci, but still less than CHX. Keyword: Mutans Streptococci, Small Cardamom, Chlorhexidine, De-ionized water. (J Bagh Coll Dentistry 2013; 25(4):160-163). الخالصة لجنس ولھ تاریخ قدیم قدم ا, الھیل األخضر ھو ملك التوابل لما لة من راحة عطرة جدا وطعم.یجري استكشاف اإلعشاب على نطاق واسع كبدائل الصناعیة مضادة للبكتریا : المقدمة الغرض من ھذه الدراسة دراسة تأثیر تراكیز مختلفة من المستخلص المائي والكحولي للھیل على . معظم الناس یستخدمون الھیل كتوابل وغیر مدركین فوائدة الصحیة . البشري .وااللتصاق لبكتریا المكورات المسبحیة مختبریا, النمو, الحساسیة شملت التجربة اختبار حساسیة المیوتانز للتراكیز المختلفة لمستخلص الھیل .)22-18(تم جمع اللعاب من عشرة طالب تتراوح أعمارھم بین , في ھذه الدراسة :المواد والعمل كذلك تم دراسة تاثیر مستخلص الھیل على النمو الحیوي للمیوتانز وعلى قابلیة البكتریا لاللتصاق , ن الحفر في الوسط البكتیرياألخضر والمواد الضابطة األخرى بطریقة االنتشار م .على األسنان كلورھكسدین % 0.2لكن یبقى تاتیرھما اقل من . كان المستخلصان المائي والكحولي فعالن في تثبیط ھذه البكتریا, حسب طریقة االنتشار من الحفر في الوسط البكتیریا مختبریا :النتائج %) 20و% 15و% 10(تم اختبار تاثیر تراكیز . (P<0.01) .المستخلص الكحولي كان لھ تأثیر أقوى من المستخلص المائي بنفس التراكیز بفروق إحصائیة عالیة .كلوكونیت وجد آن مستخلص الھیل المائي والكحولي و الكلورھكسدین لھ فروقات إحصائیة عالیة في تقلیل النمو الحیوي و, للمستخلص المائي والكحولي على النمو الحیوي للمیوتانز مختبریا د ان وج .كلورھكسدین كلوكونیت% 0.2لكن یبقى المستخلص المائي والكحولي للھیل اقل فعالیة من .ساعة 24للبكتریا مقارنة بالنمو الحیوي للبكتریا بدون إضافة إي عامل بعد مرور كلورھكسدین كلوكونیت فعال في منع االلتصاق % 0.2 جمیع التراكیز المستخدمة لمستخلص الھیل المائي و الكحولي مختبریا غیر فعالة في منع التصاق البكتریا على األسنان وان .البكتریا .رھكسدین كلوكونیتأن مستخلص الھیل كان فعاال ضد بكتریا المیوتانز ولكن اقل تأثیرا من كلو: االستنتاج .الماء الغیر ایوني, الكلورھكسدین كلوكنیت, الھیل األخضر, المكورات المسبحیة المیوتانز :كلمات مفتاحیھ INTRODUCTION Dental caries is one of the most common infectious diseases in oral human cavity (1,2). The mouth contains a wide variety of oral bacteria, but only a few species of bacteria are believed to cause dental caries; Mutans streptococcus and Lactobacilli (3-5). The elimination of cariogenic bacteria from the oral cavity using antibacterial agents is one of primary strategies for prevention of dental caries (6). Medicated oral rinses usually contains antimicrobial agents, such as chlorhexidine gluconate which is very potent chemo-prophylactic agent, it has abroad spectrum action especially against Mutans streptococci (1) MSc student, Department of Pedodontics and Preventive dentistry, College of Dentistry, Baghdad University (2) Professor, Department of Pedodontics and Preventive dentistry, College of Dentistry, Baghdad University group. But it has many side effect like staining of teeth, altering the test of the mouth and desquamation of oral mucosa (7,8). Herbs are being widely explored to discover alternatives to synthetic antibacterial agents (9). Small cardamom often referred to as queen of spices because of its very pleasant aroma and taste, has a history as old as human race (10). Most people use cardamom as a spice and are largely unaware of its numerous health benefits. In addition to its wide use for culinary purpose, cardamom has folkloric repute as carminative, stomachic, diuretic, antibacterial, analgesic, antiviral, anti-inflammatory, antifungal and is considered useful in treatment of many diseases (10,11). There are very little exclusive studies about small cardamom antibacterial effect on dental caries. For all of the above this study was conducted. J Bagh College Dentistry Vol. 25(4), December 2013 Effect of small cardamom Pedodontics, Orthodontics and Preventive Dentistry161 MATERIALS AND METHODS Small cardamom fruits were obtained from AL-Shoorga market. Small cardamom fruits were American origin grade 4. The samples were carefully washed under de-ionized followed by sterile distilled water and then air dried for two days, pounded using a mixer grinder and stored in air tight bottles. There are two methods for extraction: water extraction and alcoholic extraction. For water extract 100grams of fruit powder of cardamom was soaked in 1000ml cold sterile distilled water in a conical flask and left undisturbed for 24h. For alcoholic extract 100 grams of fruit powder was kept in 70% ethanol for 3 consecutive days at room temperature. Then both extract filtered off using a sterile Whatman filter paper No1 (12).The filtered extract was concentrated under vacuum below 40oC using a rotaevaporator. The weight of the solid residue was recorded and taken as the yield of crude extract (13). Stimulated saliva was collected from ten healthy looking students from University of AL-Mustansiriya aged (18-22) years in order to carry out in vitro experiments from which Mutans streptococci were isolated, purified, and diagnosis according to morphological, microscopical, biochemical test and by VITEK2 test. Agar well technique was applied to study the antibacterial effects of different concentrations of water and alcoholic cardamom extracts (5%, 10%, 15%, 20%, 25%, 30%), compared with chlorhexidine 0.2% as a control positive and de- ionized water as control negative on MHA media. These experiments were conducted on 10 isolates of Mutans streptococci. The viability counts of Mutans streptococci inoculated from broth media, to which 10%, 15%, and 20% of water and alcoholic cardamom extracts, CHX 0.2% and de-ionized water were added have been estimated in comparison to the control (broth and bacteria only).The procedure was carried on 5 isolates of Mutans streptococci. The prevention of adherence of Mutans streptococci to the teeth and stainless wire after the 10%, 15%, and 20% of water and alcoholic cardamom extracts, chlorhexidine 0.2% and de-ionized water compared to the control positive (broth and bacteria without agent) and control negative (broth and agent without bacteria) had been tested in vitro These experiments carried on 50 extracted first premolars (right and left sides) form Orthodontic department. RESULTS Sensitivities of Mutans streptococci (MS) to different concentrations of cardamom, CHX and de-ionized water in vitro were determined by using agar well diffusion method. The diameter of inhibition zone (clear zone of no growth of MS around each well) was found to increase as the concentrations of cardamom extracts increase. De-ionized water showed no zone of inhibition while CHX showed the highest zones of inhibition compared to the cardamom extracts as shown table (Table 1).Alcoholic extracts showed higher zone of inhibition compared to the same concentration of water with high significance differences (P<0.01) (Table 2). The counts of MS were tested in vitro in the presence of 10%, 15%, and 20 % of water and alcoholic extracts of cardamom, CHX, de-ionized water and control. LSD test used to compare the initial count, the counts of bacteria after 24 hr and their counts after using different agents. The result showed high significance differences between agents except a significance difference between initial count and CHX and no significance difference between counts after 24and de-ionized water (Table 3). All the concentrations of cardamom extracts tested were failed in the prevention of adherence of Mutans streptococci, while control negative and teeth treated with CHX showed no accumulation of dental plaque on them after seen days of incubation (Table 4). DISCUSSION Sensitivities of Mutans streptococci to different concentrations of water and alcohol extracts of cardamom by agar well diffusion method had been tested in this study. Results showed that cardamom extracted by water and alcohol were able to inhibit the growth of Mutans streptococci, this finding were in coincidence with other studies (10,14). The diameter of zones of inhibition of MS were increased as the concentration of both cardamom extracts increased from 10% to 30% but still lower than CHX 0.2%. For alcohol extract the zones of inhibition was much higher than water extract with highly significant differences, (this finding may be explained by the fact that, the components of cardamom that had antibacterial effects against MS and inhibit its growth, and were more soluble in alcohol than water. By laboratory analysis of small cardamom by HPLC (High-performance liquid chromatography), in this study, it was found that concentration of major active compounds (1-8 cineole, α-terpinyl acetate) is higher in alcohol extract than in water extract. The antimicrobial property of small cardamom has been shown to be attributable to the essential oil fraction (15). J Bagh College Dentistry Vol. 25(4), December 2013 Effect of small cardamom Pedodontics, Orthodontics and Preventive Dentistry162 A highly significant reduction was found in the viable counts of Mutans streptococci in 10%, 15% and 20% of both cardamom extracts compared to the control after 24 hr. It could be attributed to chemical constituents of small cardamom like Cineole; the major active component of cardamom oil. It is a potent antiseptic that is known to kill bacteria producing bad breath and other infections (10). No one of any concentration of water and alcohol cardamom mouth washes tested was able to prevent adherence of bacteria. However in comparison to the control, plaque thickness was less. This reduction in plaque thickness may be attributed to the inhibitory effect of these agents on growth or metabolism of these bacteria rather than on adherence ability. Table 1: Mean and SD of MS inhibition zone in millimeter to different concentration to different agents (Agar well diffusion methods) Agents No. Mean ± S.D ANOVA test CHX 10 17.50 0.57 F=349.541 d.f=10 P=0.000 HS Water extract 5% 10 0 0 Water extract 10% 10 7.35 0.66 Water extract 15 % 10 9.10 0.45 Water extract 20 % 10 10.15 0.52 Water extract 25 % 10 11.20 0.34 Water extract 30 % 10 12.55 0.49 Alcoholic extract 5 % 10 0 0 Alcoholic extract 10 % 10 10.35 0.62 Alcoholic extract 15% 10 12.20 0.63 Alcoholic extract 20% 10 14.75 0.48 Alcoholic extract 25% 10 16.20 0.78 Alcoholic extract 30% 10 16.90 0.51 De-ionized water 10 0 0 Mean (mm). Table 2: LSD test between sensitivity of Mutans streptococci to same concentration of both water and alcoholic extracts of cardamom (Agar well diffusion method) Water extract Alcoholic extract concentration Mean Difference P-value Description 10% -3.00 0.000 HS 15% -3.10 0.000 HS 20% -4.60 0.000 HS 25% -5.00 0.000 HS 30% -4.35 0.000 HS Table 3: LSD between agents in comparison with initial counts and counts after 24 Agents Initial Count Count After 24 Mean Difference P-value Description Mean Difference P-value Description W.E 10% 172.6 0.000 HS -82.0 0.000 HS W.E. 15% 138.4 0.000 HS -116.2 0.000 HS W.E. 20% 94.2 0.000 HS -160.4 0.000 HS A.E. 10% 150.8 0.000 HS -103.8 0.000 HS A.E. 15% 104.0 0.000 HS -150.6 0.000 HS A.E. 20% 56.4 0.004 HS -198.2 0.000 HS D.W. 222.0 0.000 HS -32.6 0.114 NS CHX -45.4 0.018 S -300.0 0.000 HS W.E= Water extract A.E= Alcoholic extract D.F=8 J Bagh College Dentistry Vol. 25(4), December 2013 Effect of small cardamom Pedodontics, Orthodontics and Preventive Dentistry163 Table 4: The effects of cardamom, de-ionized water and CHX on adherence of MS in vitro Agents (2 minutes) Adherence Control positive +ve Control negative -ve 10 % water cardamom extract 15% water cardamom extract 20% water cardamom extract +ve +ve +ve 10% alcoholic cardamom extract 15 % alcoholic cardamom extract 20 % alcoholic cardamom extract +ve +ve +ve CHX -ve De-ionized water +ve REFERENCES 1. 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