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Indian Journal of Natural Products and Resources Vol. 3(1), March 2012, pp. 123-127 Short Communications Efficacy of some medicinal plants against human dental pathogens Anupama Pathak*, Aparna Sardar, Vynkatesh Kadam, Bhagwan Rekadwad and S Mohan Karuppayil School of Life Sciences SRTM University, Nanded- 431 606, Maharashtra, India Received 2 June 2011; Accepted 2 November 2011 Dental caries is one of the most prevalent diseases having multifactorial etiology. Most of the treatments in dental caries is aimed at either elimination or suppression of bacteria by antibiotics. Increased resistance of oral bacteria to antibiotics however, has developed keen interest of researcher in herbal treatment. In our investigation we have screened nine medicinal plants from Marathwada region. We have proved effective inhibition of dental pathogens by aqueous extract of plants. Amongst selected plants leaves of Emblica officinalis Gaertn. syn. Phyllanthus emblica Linn. and Terminalia bellirica Roxb. and flowers of Syzygium aromaticum (Linn.) Merrill & Perry showed three to four times more inhibition in comparison with effective antibiotics like Carbenicillin, Tetracycline, Nalidixic acid and Nitrofurantoin of four dental pathogens, viz. Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis and Actinobacillus actinomycetemcomitans isolated by us. Keywords: Dental pathogen, Dental caries, Medicinal plants, Emblica officinalis, Terminalia bellirica, Syzygium aromaticum IPC code; Int. cl. (2011.01)—A61K 36/00, A61P 31/04 Introduction Dental caries is a very common problem in humans1. It is very prevalent in Asian and Latin countries and least prevalent in African countries. In India, nearly 60-70% of the child population is affected by dental caries2. The main etiological agents of dental caries are Streptococcus mutans, S. mitis, S. sanguis, S. salivarius, and S. sobrinus. Fermentation of carbohydrate by acidogenic oral bacteria is the key factor in the development of dental caries. The acid released through microbial action leads to demineralization and cavitations of tooth3. S. mutans can colonize the tooth surface and initiate plaque formation by synthesizing extracellular polysaccharide _____________ *Correspondent author: E-mail: [email protected]; Phone: 02462 229242; Fax: 02462-229245 from sucrose. Accumulation of plaque around the gingival margin and subgingival region may lead to shifts in the population of the microflora from Gram positive to Gram negative bacteria1. These Gram negative plaque forming bacteria may cause periodontal disease in periodontal tissues and alveolar bone surrounding the teeth. Several potential periodontal pathogens have been studied and Gram negative anaerobic bacteria such as Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans and Camphylobacter rectus are considered to represent a significant portion of pathogenic bacteria1. Dental caries are generally treated with effective antibiotics like Chlorohexidine digluconate, Penicillin, Methicillin, Ampicillin, Erythromycin and Cephalothin. However, it was observed that herbal products used against oral infection have more inhibitory effect on dental pathogen. Various plants and extracts are used traditionally for dental care. Twigs of plants like Babul, Neem, clove oil and many others are used for brushing teeth in India3-4. In this study, we are reporting the efficacy of aqueous extracts of nine medicinal plants against four pathogens causing dental caries. Materials and Methods Preparation of aqueous extract Plants were collected from Nanded, District of Maharashtra, and identified with the help of regional flora; powdered and stored at 4°C throughout investigation. Few plants were purchased in powder form from the local market. Dried leaves of Azadirachta indica A. Juss., Acacia nilotica (Linn.) Delile subsp. indica (Benth.) Brenan, Ocimum basilicum Linn., Emblica officinalis Gaertn., Hemidesmus indicus R. Br., Terminalia bellirica Roxb., Syzygium cuminii (Linn.) Skeels and flowers of Spilanthes calva DC. and Syzygium aromaticum (Linn.) Merrill & Perry were used to prepare aqueous extract. Botanical and common names along with their family and parts used are given in Table 1. The aqueous extract of plant material was prepared by using a Soxhlet apparatus. The extracts were filtered through a musclin cloth. Filtrate was then INDIAN J NAT PROD RESOUR, MARCH 2012 124 Table 1Plants used in this study Sample No. Scientific name 1 2 5 3 4 6 7 8 9 Acacia nilotica (Linn.) Delile subsp. indica (Benth.) Brenan Azadirachta indica A. Juss. Emblica officinalis Gaertn. Hemidesmus indicus R. Br. Ocimum basilicum Linn. Spilanthes calva DC. Syzygium aromaticum (Linn.) Merrill & Perry Syzygium cuminii (Linn.) Skeels Terminalia bellirica Roxb. concentrated using reflux method at 100°C for six hours in Soxhlet apparatus to one fourth of the original volume and used5- 11. Isolation of bacteria Ten samples were collected from a dental clinic and civil hospital at Nanded. Isolation of bacteria was carried out using blood agar and nutrient agar. Typical colonies were selected after sufficient growth and subcultured on nutrient agar. Various biochemical tests were performed to identify clinical isolates. Isolates were identified using Bergey’s manual of Systematic Bacteriology112-13. The clinical isolates were inoculated into nutrient broth (Hi-media Pvt limited, Mumbai, India) and incubated at 37°C for 24 h and the suspension was checked to provide 105 CFU/ml by dilution plate count method4, 6. Antibacterial assay Antibacterial assay was performed using agar well diffusion method4. Plates were prepared and 0.1ml of culture broth was added and spread with a sterile spreader. A well was made in the centre of plate with the help of a cork borer (0.65 cm). 100µl test compound was introduced into the well and the plates were kept in a refrigerator for diffusion for 30 min and then incubated overnight at 37°C. The antimicrobial activity was interpreted by measuring the diameter of zone of inhibition in mm. Sensitivity of the clinical isolates to six commonly used standard antibiotics, viz. Ampicillin 25mcg, Gentamycin 10 mcg, Carbenicillin 10 0 mcg, Nalidixic acid 30 mcg, Nitrofurantoin 50 mcg, Tetracycline 200 mcg per disc (Hi-media Pvt, Mumbai, India) was evaluated. All experiments were performed in the triplicates. Results and Discussion Four different types of organisms were isolated from ten different clinical samples. Morphological Family Common name Part used Fabaceae Kikar Foliage Meliaceae Euphorbiaceae Asclepiadaceae Lamiaceae Asteraceae Myrtaceae Myrtaceae Combretaceae Neem Awala Anantmul Tulsi Akkal khara Clove Jambhul Behada Foliage Foliage Foliage Foliage Floral parts Floral parts Foliage Foliage and biochemical characters were studied performing different tests (Table 2). Identification of the isolates was carried out as per Bergey’s Manual of systematic bacteriology the organisms were S. mitis, S. sanguis, S. mutans and A. actinomycetemcomitans. Out of nine, eight plants inhibited the growth of S. mitis. The inhibitory activity was comparable to that of standard antibiotics. Among these extracts tested, E. officinalis was most effective followed by T. bellirica. The activities of these extract were twofold more than that of the standard antibiotics. The order of inhibition was as follows: E. officinalis > T. bellirica > S. cuminii > S. aromaticum > A. indica > A. nilotica subsp. indica > O. basilicum > S. calva (Table 3). S. sanguis was inhibited by seven plant extracts in above sequence except S. calva which showed no activity against S. sanguis. The antimicrobial activity was 2.5 times more than the standard antibiotics. (Plate 1). Eight plant extracts showed remarkable inhibition of S. mutans. T. bellirica extract showed highest inhibition and it was three times more as compared to Carbenicillin, Nalidixic acid and Nitrofurantoin. Other eight extracts mentioned above also inhibited S. mutans effectively. All the nine plant extracts showed inhibition of A. actinomycetemcomitans. T. bellirica was more effective followed by S. cuminii and E. officinalis. Overall three times more inhibition was observed with plant extracts when compared with standard antibiotics. The order of inhibition was as follows T. bellirica > S. cuminii > E. officinalis > S. aromaticum > A. nilotica subsp. indica > A. indica > S. calva > H. indicus > O. basilicum (Plate 1). It is very common practice to use antibiotics like Penicillin, Methicillin, Ampicillin, CHX, Erythromycin and Cephalothin against bacteria SHORT COMMUNICATIONS 125 Table 2Morphological and biochemical characteristics of clinical isolates Character Morphological and biochemical characteristics DC-1 DC-2 DC-3 DC-4 Pigmentation Pale Yellow Yellow Pink White Colony shape Circular Circular Circular Circular Colony diameter 2 mm 3 mm 1 mm 1 mm Elevation Flat Flat Elevated Elevated Margin Entire Entire Entire Entire Opacity Opaque Opaque Opaque Opaque Morphology Cocci Cocci Cocci Rod Consistency Moist Moist Moist Moist Gram’s staining Gram positive Gram positive Gram positive Gram negative Motility Non motile Non motile Non motile Non motile Starch hydrolysis + + + + Urease production _ _ _ _ Gelatin hydrolysis _ _ _ _ Glucose utilization + + + _ Sucrose utilization + _ + _ Xylose utilization _ _ _ _ Sorbitol utilization _ _ _ _ Catalase test + + _ _ H2S production _ _ _ _ Indole production + + + + Methyl red test + _ + + Voges proskauer test + + _ _ Citrate utilization + + + + Identified as S. mitis S. sanguis S. mutans A. actinomycetecomitans Table 3Antibacterial activity of water extract plants and some of the standard antibiotics against bacteria isolated from dental caries Plant Extract/Antibiotics Spilanthes calva Azadirachta indica A. Juss. Acacia nilotica (Linn.) Delile subsp. indica (Benth.) Brenan Ocimum basilicum Linn. Syzygium aromaticum (Linn.) Merrill & Perry Emblica officinalis Gaertn. Hemidesmus indicus R. Br. Terminalia bellirica Roxb. Syzygium cuminii (Linn.) Skeels Ampicillin 25 mcg/disc Gentamycin 10 mcg/disc Carbenicillin 100 mcg/disc Nalidixic acid 30 mcg/disc Nitrofurontoin 30 mcg/disc Tetracyclin 200 mcg/disc S. mitis 10.3±0.57 11.6±0.57 11±0.57 10.6±0.57 19.3±0.57 24.6±0.57 23±00 19.3±0.57 11.3±0.5 11.3±0.5 11.6±0.57 10.3±1.7 11±00 10.3±1.7 causing dental caries. As these antibiotics are active only against planktonic bacteria, more antimicrobials are now screened for their effect against oral bacteria3. Use of plant and plant products are good alternative to antibiotics. Plant product like clove oil, fresh twigs of babool and neem, etc. are used by Zone of inhibition in mm S. sanguis S. mutans A. actinomycomitans 0±00 11.6±0.57 13±1.0 10.6±1.1 13.6±0.57 16.6±0.57 11.3±1.1 19.3±0.57 20.6±0.57 10.6±0.57 14±1.00 10.3±0.57 23.6±1.15 00±00 28.6±1.15 29.6±0.57 29.6±0.57 32.6±0.57 00±00 10.3±0.57 11±0 28.6±1.1 33.6±1.1 36.3±2.3 25±00 26.3±1.15 33.6±1.15 10.6±1.1 10.0±0.0 12.3±1.1 12±00 12.3±1.1 10.6±1.1 11.3±1.15 13±1 12.3±0.57 12.6±0.5 11±00 12±2 0±00 10.6±0.57 10.6±1.1 11.3±0.5 10±00 12.3±1.1 villagers in India and also they have been validated by various research findings14-15. Many popular tooth pastes contained extracts of plants which include clove, menthol, neem and triphala2. Triphala which is the mixture E. officinalis, T. bellirica and T. chebula is widely used as a mouthwash and for mouth ulcers. 126 INDIAN J NAT PROD RESOUR, MARCH 2012 Plate 1 Inhibitory effect of aqueous extract of selected plants on dental pathogens SHORT COMMUNICATIONS Conclusion Based on results it is concluded that T. bellirica, E. officinalis and S. aromaticum possess antimicrobial activity against pathogens causing dental caries. Selected plant extracts are 2-3 folds more effective than common antibiotics and these plants are very safe and have acceptable taste. References 1 Kanso Iwaki, Satomi Koya-Miyata, Keizo Kohno, Shimpei Ushio and Shigeharu Fukuda, Antimicrobial activity of Polygonum tinctorium Lour: extract against oral pathogenic bacteria, J Nat Med, 2006, 60, 121-125. 2 Shobha Tandon, Kunal Gupta, Sugandhi Rao and KJ Malagi, Effect of Triphala mouthwash on the caries status, Intern J Ayur Res, 2010, 1(2), 93-99. 3 Barira Islam, Shahper N. Khan, Asad U. 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