Journal of Oral Health ORIGINAL ARTICLE Community& Dentistry An In Vitro Microbiological Study Evaluating the Efficacy of Soluneem (A Water Soluble Neem Formulation from Azadirachta Indica) Against Periodontopathic Microorganisms Shefali Sharma1, Suchetha A2, Vijayendra R3, Bharwani Ashit G4 ABSTRACT Objectives: To evaluate the efficacy of Soluneem (a water soluble formulation from the neem seed kernel from Azadirachta Indica containing Azadirachtin) as an antimicrobial agent and the effective concentration of Soluneem required to inhibit periodontopathic bacteria and to compare it with a known antiplaque agent chlorhexidine (0.2%) in vitro. Material and Methods: The subgingival plaque samples from a total of 20 sites from 5 patients (4 sites per patient) was taken with a probing pocket depth of >”5mm. Subgingival plaque samples were collected, cultivated, and incubated anaerobically as per the standard procedure. Further subculturing was done to obtain pure growth. Various biochemical tests were used for identifying each organism as per the standard protocol. The sensitivity to the varying concentrations of neem like 100%, 50%, 25%, 12.5%, 6.25%, 3.125% and 1.5625% and 0.2% Chlorhexidine solution was tested using the Modified Disc diffusion method. The sensitivity was tested by measuring the zones of inhibition comparing with the known standard diameter. Results: The organisms isolated and percentage of identification rate from total of five patients was 60% for Bacteroids fragilis, 40% for Bacteroids distasonis, 20% for Prevotella corporis, 20% for Prevotella melaninogenica, and 20% for Peptostreptococus species. Chlorhexidine 0.2 % was effective against micro organisms cultivated and isolated in this study. However Soluneem was not effective against the organisms cultivated in this study. Conclusion: Soluneem had no effect on the growth of any of the isolated organisms. Further studies testing soluneem against other putative periodontal pathogens with Azadirachtin are warranted. Keywords: Azadirachtin, Microbial sensitivity testing, Soluneem, Zone of inhibition. INTRODUCTION 1M.D.S Senior Lecturer 2M.D.S, Professor and Head Dept. of Periodontia Dept. of Periodontics he development of chemothera Seema Dental College and Hospital, D.A. Pandu Memorial R.V. Dental College, peutic agents capable of inhibiting Rishikesh, India Bangalore, India Tdental plaque formation has been 3M.D.S, Former Head of Department, of great interest to dental researchers and Contact Author Dept of Periodontics clinical dentists over the past decade (1). In D.A. Pandu Memorial R.V. Dental College, Bangalore, India the recent years, human pathogenic micro- Dr. Bharwani Ashit G organisms have developed resistance in re- [email protected] 4Post Graduate student, Dept. of Periodontics D.A. Pandu Memorial R.V. Dental College, sponse to the indiscriminate and long term J Oral Health Comm Dent 2012;6(1)4-9 Bangalore, India use of antimicrobial drugs commonly 4 JOHCD www.johcd.org January 2012;6(1) AN IN VITRO MICROBIOLOGICAL STUDY EVALUATING THE EFFICACY OF SOLUNEEM employed in the treatment of infectious Earlier preparations from the Neem seed biotic treatment for any purpose in the past diseases. The undesirable side effect of cer- kernel were available as emulsified concen- 6 months including the use of mouth- tain antibiotics, and the emergence of previ- trates and required many solvents. These wash were excluded from the study. ously uncommon infections, has made the degrade very quickly in the presence of wa- scientists to look for new antimicrobial sub- ter or sunlight. Soluneem is highly stable STUDY DESIGN stances of alternate sources like plant origin. even at 1100C, whereas technically Neem The study was carried out using 20 sub- Therefore plants represent a potential source Seed Kernel Extracts (NSKE) is thermola- gingival plaque samples from patients di- of new anti-infective agents (2). bile. The unique advantage of this prepa- agnosed with chronic periodontitis. The Indians have used herbal products for oral ration is that it is a water soluble formula- subgingival plaque samples from peri- care for centuries. Most common ones be- tion which will have, a potentiated action odontal pockets >” 5mm were collected. ing extracts of Mango (Mangifera indica), and it contains the compound azadirachtin Supragingival plaque was removed with Neem (Azadirachta indica; Melia azadirachta), (9). hand instruments and the subgingival and Tea-dust (Camellia sinensis) (3).The plaque was collected by inserting a steri- most widely used herbal product among The insecticidal activity of this product has lized Gracey curette (Area-specific curette) them is Neem and its products. The Neem been confirmed and it has been suggested into the deepest portion of the periodon- twig has been used from time immemorial that it may have other applications in the tal pocket (13). for cleansing the teeth and the juice extracted field of personal care. In the present study by it’s chewing was believed to be beneficial an attempt has been made to explore the The plaque samples collected were plated for toning the gums (4).The widespread use effective concentration of Soluneem re- immediately onto 5% sheep blood agar, of the Neem “chewing stick” as an oral hy- quired to inhibit periodontopathic micro- Brucella Blood Agar (BBA) and Kanamy- giene device in certain Asian and African organisms and to compare Soluneem with cin Vancomycin Laked Blood Agar countries has suggested that it may possess antibacterial efficacy of chlorhexidine which (KVLBA) using sterile nichrome wire loop some anti-plaque properties also. Dentists is considered as the gold standard in peri- (14).This was immediately transferred into in areas where it is in use , have noted its odontics (12). an anaerobic jar with gas pack system (Fig- effectiveness in preventing certain plaque- ure-1). The sheep blood agar plates were related dental diseases (1). MATERIALS AND METHODS incubated aerobically at 37oC for 72 hrs; Patients diagnosed with chronic periodon- BBA and KVLBA were incubated anaero- Several studies have reported the antibac- titis and attending the Department of Peri- bically at 37 oC for 72 hours. terial effects of chewing sticks on odontics, D.A.P.M.R.V. Dental College, periodontotal pathogens particularly Bangalore, formed the study group. The gram stain smear from each patient Bacteriodes species and inhibitory action was examined for the presence of bacteria on dental plaque formation (1,4,5,6). Patients were explained about the purpose and pus cells (15) (Figure-2). Individual of the investigation and a written consent colony morphotypes were subcultured The quest for identifying the various was taken prior to commencement of the onto Chocolate Agar (aerobic inoculation chemotherapeutic agents in neem has led study. Ethical clearance was obtained by the for aerotolerance) and Brucella Blood Agar to the isolation of more than 135 com- ethical committee of the institution. enriched with Vitamin-K and Heame for pounds. The compounds have been di- obtaining pure growth. These were fur- vided into two major classes: isoprenoids Commercially available Chlorhexidine with and others. The isoprenoids includes a concentration of 0.2% (w/v) was selected azadirachtin (7). for the study to be compared for its efficacy with Soluneem. Azadirachtin has been found to inhibit enzymatic activity of larvae of Drosophila INCLUSION CRITERIA melanogaster, adult female Aedes aegypti, Patient aged 30-55 years with a diagnosis the larvae of Manduca sexta(8) and black- of chronic periodontitis; free from systemic headed caterpillar (9).The Neem seed ker- disease and who had not undergone any nel extracts and oil containing azadirachtin form of non surgical/surgical periodontal has shown antibacterial activity and activity therapy in the last 6 months were included against dermatophytes (10,11).One such in the study. example of a compound containing azadirachtin is SoluneemTM. Soluneem is a EXCLUSION CRITERIA water soluble formulation from the neem Patient with a known systemic disorder, seed kernel from Azadirachta Indica con- pregnant women, smokers, and patients Figure 1: Anaerobic jar with gas taining Azadirachtin –A 6%. who received any topical or systemic anti- pack system JOHCD www.johcd.org January 2012;6(1) 5 AN IN VITRO MICROBIOLOGICAL STUDY EVALUATING THE EFFICACY OF SOLUNEEM zone of inhibition was >” 6.1 mm colonies; few black pigmented colonies on (18).The standard strain ATCC Bacteroides Kanamycin Vancomycin laked Blood agar fragilis was included for biochemical reac- were also seen. The anaerobes identified tions and sensitivity testing (14). were- Prevotella corporis, Prevotella melaninogenica, Bacteroids distasonis, Bacteroids RESULTS fragilis, and Peptostreptococus species. Dental plaque inoculated from the sam- ples gave a confluent growth with varied The percentage of identification rate from TABLE 1: BIOCHEMICAL TESTS Table 1.1: Biochemical tests of Prevotella corporis (+) → Positive and (-) → Negative Prevotella Corporis Figure 2: Gram stain smear to Indole Nit Bile Broth Glucose Sucrose Malt detect presence of bacteria and pus cells ññResistant + + + Lactose Arabinose Rhamnose Trehalose Salicin Esculin ther identified using standard biochemical ññññññ tests which included: Indole production, - Nitrate reduction, Aesculin hydrolysis, Table 1.2: Biochemical tests of Prevotella melaninogenicas Growth in Bile, Fermentation Tests–Glu- (+) → Positive and (-) → Negative cose, Sucrose, Malt, lactose,