You are on page 1of 3

Hari Prasad O et al.

/ Journal of Pharmacy Research 2011,4(7),2149-2151

Research Article ISSN: 0974-6943

Available online through

Comparative evaluation of the antibacterial efficacy of P. juliflora and three commercially available mouthrinses: an in vitro study
Hari Prasad O *1, Sudheer Aluru 1, Kishore Kumar A 2, Navya A 3, Hari Krishna O 2, Bhaskar M 1, Papa Rao A 4, Ravindra Reddy N 2 1 Division of Animal Biotechnology, Department of Zoology, S.V University, Tirupati, Andhra Pradesh, India 2. Department of Periodontology, CKS Teja Dental College,Tirupati, Andhra Pradesh, India 3. Department of Applied Microbiology, SPMVV, Tirupati, Andhra Pradesh, India 4. Department of Anthropology, S.V University, Tirupati, Andhra Pradesh, India

Received on: 12-04-2011; Revised on: 18-05-2011; Accepted on:21-06-2011 ABSTRACT

Oral and periodontal infections are common in normal population and high in immunocompramised patients. These diseases affect the tissues surrounding teeth, generally caused by insufficient oral hygiene, leading to plaque formation, harboring several pathogens. There are several synthetic and chemical compounds available in market, promising to be effective but it is well known that on prolonged use, these chemical compounds produce side effects, so as an alternative leaf extract of Prosopis juliflora is used. The objective is to screen and compare the antibacterial activity of P. juliflora crude extract with commercially available mouthrinses on oral and periodontal organisms. Antibacterial activity was determined by MIC and results were calculated using Instat plus. The activity of P. juliflora was observed to be highest in comparison with the other commercial mouthrinses against selected microbes. The present results clearly indicate the crude extract of P. juliflora is an effective antibacterial agent against oral and periodontal microorganisms compared to commercially available three mouthrinses. Key words:Prosopis juliflora , mouthrinse, periodontal infections, antibacterial activity.

Periodontal diseases represent possible reservoirs of opportunistic bacteria in the oral cavity, which display important virulence properties and cause a wide range of human systemic diseases including pneumonia, septicemia, and endocarditis [1,2,3,4]. The microbiota associated with localized aggressive periodontitis is composed of gram negative, capnophilic and anaerobic bacteria [5,6,7]. Enterococcus faecalis is the most frequently isolated species from endodontic infections and associated with re-infection leads to subsequent failure of endodontically treated teeth [8,9,10]. Anaerobes too play a predominant role in most periodontal infections. Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola and Aggregatibacter actinomycetemcomitans are some of the organisms which are found in periodontal infections [5]. A modest association was also observed between periodontitis and cardiovascular disease, stroke and atherosclerosis [11]. Mouthrinses are most frequently recommended to patients whose mechanical oral hygiene procedures are inadequate for the plaque control. This study provides an additional rationale of mouthrinses in effective control of oral bacteria. The success of any mouthrinse depends on its ability in inhibiting or killing a varied number of pathogenic oral and periodontal microflora. Chlorhexidine, the most commonly used compound in various mouthrinses is a proved antimicrobial agent. This is used in combination with a variety of compounds under different brand names. But prolonged use of Chlorhexidine can cause several side effects including staining of teeth, gastro-intestinal problems, gingivitis, dry mouth and many more [12]. There are many natural ways to treat periodontal diseases. Herbal remedies like Aloe vera, Clove oil have been reported to relieve pain and discomfort when applied on gums [13]. Herbal remedies have an edge over conventional antibiotics which suffer the limitation of low benefit to high risk as compared to herbal treatment which possess high benefit to low risk ratio [14]. Prosopis juliflora , a convenient neighborhood plant has a wide range of applicability since ages as a folk medicine. Pharmacological properties under in vitro conditions shows that it has antibacterial [15,16,17,18], antifungal [19,20], hemolytic [21] anti-inflammatory [20] and wound healing activities. Antitumor activity against several human epithelial and hepatic tumor cells [20] and MDA-MB-231 breast adenocarcinoma cells [22] was also recorded. It is known from earlier literature that P.juliflora leaf extract contains flavonoids Apigenin and Quercetin which shows antibacterial activity [23]. It also used in healing bladder infections, painful gums and headaches [24,25]. The present study evaluated the in vitro antibacterial efficacy of P. juliflora leaf crude extract against three commercially available and popular mouthrinses. The selected microbes S. aureus, E. faecalis, A. actinomycetemcomitans, P. intermedia and P. gingivalis were pathogenic that predominantly involve in oral and periodontal infections causing bleeding on probing and increased pocket depth [25]. They have significant relation in Cardio Vascular Diseases (CVDs), endothelial damage and cholesterol plaque formation [26]. MATERIALS AND METHODS Preparation of Plant extract: P. juliflora leaves were selected from in and around Tirupati and Taxonomical identification of the plant was done by department of Botany, Sri Venkateswara University, Andhra Pradesh, India. Care was taken while selecting the leaves to be free from any disease. Leaf samples of 100g were surface sterilized with autoclaved distilled water, twice. These were allowed to air dry and blotted for any excess water. This was now macerated with 100ml sterile distilled water and blended for 10 minutes to obtain a homogenous mixture. Filtration of this mixture with double layered muslin cloth and subjecting it to 4000g centrifugation for 30 minutes gave a supernatant which on filtration with Whatmann No.1 filter paper gave a pure solution [27]. This served as aqueous extract of the plant and 1g/ml of the obtained extract was used as stock solution labeled D. Collection of Mouthrinses: Three commercially available mouthrinses with different chemical combinations were taken as A, B and C. Mouthrinse A was chemically composed of Chlorhexidine Gluconate (0.2% w/v), Sodium Flouride IP (0.05% w/v) and Zinc Chloride IP (0.09% w/v). Mouthrinse B had Chlorhexidine Gluconate solution IP diluted to Chlorhexidine Gluconate (0.2% w/v). Mouthrinse C contained purified water, Sorbitol, Alcohol, n-Propanol, Poloxamer 407, Benzoic Acid, Sodium Saccharin, mouthwash Flavour, Eucalyptol, Methylsalicylate, Thymol, Sodium Banzoate and Menthol. Ciprofloxacin (CF) - 5g Disc (Hi-Media laboratories, India) was used as control.

*Corresponding author.
*Dr.Hari Prasad Osuru, Department of Pathology, University of Virginia School of Medicine, MR5 Building,415 Lane Road, Charlottesville, Virginia-220908-0904, USA. Tel : +14342422024, +14342708763.

Journal of Pharmacy Research Vol.4.Issue 7. July 2011


Hari Prasad O et al. / Journal of Pharmacy Research 2011,4(7),2149-2151

Bacterial Strains and Culture conditions: The characterized clinical microbial strains of Enterococcus faecalis, Staphylococcus aureus, Prevotella intermedia,Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were collected from Department of Periodontalogy, CKS Teja Dental College, Tirupati, Andhra Pradesh. The cultures were sub-cultured onto respective selective media. In brief, S. aureus was cultured on Baird-Parker agar; E. faecalis on Mac Conkeys Agar, BHI and TSBV were used to culture P. intermedia, P. gingivalis and A . actinomycetemcomitans (All the culture media were procured from Hi-media laboratories, India). In vitro antibacterial assay: Antibacterial activity was checked by disc diffusion method on Muller Hinton Agar (Hi-media laboratories, India) for aerobes and Wilkins Chalgren Blood Agar for anaerobes as per CLSI standards [28]. Media were prepared and poured onto Petri dishes. 0.1 ml of test organism containing 10 5 CFU/ml was uniformly spread on the surface of the media. Same procedure was followed for all the organisms [29]. After ten minutes, aseptic dried discs (dried overnight at 37 0C) containing 10l volume of each mouthrinse A, B, C and plant extract D were placed on the media. Ciprofloxacin disc (CF) of 5 g concentration was placed as control. The anaerobic plates were incubated in anaerobic jar (Hi Media Anaerobic System-Mark V, with Anaerocult gas pack, MERCK) and aerobes were incubated at 37 0C in an incubator. After the incubation time of 48h the plates were checked for their antibacterial activity by measuring the zone of inhibition surrounding the disc in each plate with Hi Antibiotic Zonescale (Hi Media laboratories) in mm. The values obtained indicated the inhibitory efficacy of mouthrinses and crude leaf extract against the test organisms. Statistical analysis: Minimum Inhibitory Concentration(MIC) was expressed in terms of Mean SD values of all six repeats for each organism against each test sample. One way ANOVA was performed using Instat + 2.02 v software. RESULTS The present results clearly indicated that P. juliflora leaf extract had maximum inhibitory activity than the commercially available mouthrinses A, B and C against test microbes which pre-dominate the oral and periodontal tissues [Figures 1, 2, 3, 4]. Sample B containing Chlorhexidine showed better activity next to plant extract. Sample C had very less activity followed by sample A. The MIC values obtained showed maximum activity of P. juliflora crude extract on E. faecalis and A. actinomycetemcomitans compared to other pathogens. Mouthrinse B had almost equal activity against E. faecalis, P. gingivalis and S. aureus. Results are showed in Table 1.
Table 1. Comparison of Antibacterial efficacy of various mouthrinses with P. juliflora leaf extract
Samples Zone of Inhibition mm (Mean SD) S. aureus E. faecalis A. actinomycetemcomitans P. gingivalis 5.000.20 10.060.30 2.200.20 10.160.28 20.230.20 4.160.15 10.090.11 2.100.10 12.200.20 22.060.30 4.000.10 12.160.20 2.230.20 12.300.30 20.000.20 5.260.25 10.000.20 3.200.20 10.060.20 24.930.30 P. intermedia 3.200.20 8.060.28 2.200.23 10.200.20 20.030.15


The p value is 0.001 indicates high significance, n = 6.

As periodontal diseases are bacterial infections, an antibacterial treatment seems to be an apt solution in controlling the disease progression; but the most common problem with conventional treatment is systemic drug administration, which give rise to toxicity issues. So as an alternative local drug administration proves to be more beneficial. Mouthrinses, irrigating solutions, dentogels and sustained release devices are some of the local delivery systems [30]. Several studies indicated the importance of mouthrinses in keeping up the oral hygiene. Hence the importance for them has been shooting up in the modern world. The active ingredient Chlorhexidine, found in most mouthrinses effectively acts against a variety of pathogens, preventing and controlling supragingival plaque and several other complications if used as per oral hygiene regimens [31,32,33,34,35,36]. Antibiotics play a vital role in control and treatment of bacterial infections. There have been several drugs formulated and practiced till date but it is known that they produce several side effects and develop resistance towards causative organisms on prolonged use. So as an alternative natural compounds such as plant extracts are used. In the present study the leaf extract of P. juliflora was tested for its antibacterial activity and minimal quantity of 100g/ 10l leaf extract was effective enough for a wide spectrum of microbes. The microbes utilized in the study were characterized clinical isolates of most potent and commonest pathogens involved with oral and periodontal infections [37]. Staphylococcus species are not usually isolated from the oral cavity and considered transitory microbiota. P. intermedia, P. gingivalis and A. actinomycetemcomitans were considered for the study as they are common pathogens of oral and periodontal infections [38]. It is known from earlier literature that the compounds like Quercetin and Apigenin have the antibacterial activity [22,23] but no work has been conducted so far against oral and periodontal organisms. This bactericidal activity of P. juliflora may be due to the presence of Quercetin having analgesic, antiallergenic, antibacterial, antidiabetic and anti-inflammatory activity and Apigenin with antiallergenic, antibacterial, antidermatitic, anti-inflammatory and antiviral activity [23]. Majority of the commercially available mouthrinses had Chlorhexidine as an active compound in form of Chlorhexidine Gluconate 0.2% w/v but the P. juliflora crude extract had no additionally added compounds still proved its antibacterial efficacy against tested organisms. Compared to mouthrinses, it had a better effect and zone of inhibition was higher than commercial mouthrinse B containing Chlorhexidine. The compounds such as Quercetin and Apigenin with well-known antibacterial activity may consider to have played a vital role in the inhibition of these pathogens too. CONCLUSION This in vitro study states the antibacterial activity of P. juliflora against oral and periodontal pathogens tested, at the outset. It was effective than the commercially available mouthrinses without any additional compounds added to it. This gives a better opportunity for further research in the concerned subject. Further purification of the extract may increase its activity proving to be more effective. However, several clinical and pharmacological trails have to be carried out for complete analysis of the active compounds in the P. juliflora leaf aqueous extract. REFERENCES
1. 2. 3. 4. Ohman SC, Osteberg Y, Dahlen G, Landahl S. The prevalence of Staphylococcus aureus, Enterobacteriaceae species, and Candida species and their relation to oral mucosal lesions in a group of 79-year-olds in Gteborg. Acta Odontol. Scand, 1995; 53: 49-54. Younessi OJ, Walker DM, Ellis P and Dwyer DE. Fatal Staphylococcus aureus infective endocarditis. Oral. Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 1998; 85: 168-72. Dahln G and Wikstrom M. Occurrence of enteric rods, staphylococci and Candida in subgingival samples. Oral Microbiol. Immunol, 1995; 10: 42-46. Van Winkelhoff AJ, Rams TR, Slots J. Systemic antibiotic therapy in periodontics. Periodontology 2000, 1996; 10: 45-78. Newman MG and Socransky SS. Predominant cultivable microbiota in periodontosis. J Periodontal Res, 1977; 12:120. Newman MG, Socransky SS and Savitt ED. Studies of the microbiology of periodontosis. J

Figure 1 - Zones of inhibition observed for Enterococcus faecalis on Muller Hinton media for Mouthrinse A, B, C, Ciprofloxacin CF and P. juliflora extract D.

Figure 2 - Zones of inhibition observed for Aggregatibacter actinomycetemcomitanson Wilkins Chalgren Blood Agar media for Mouthrinse A, B, C, Ciprofloxacin CF and P. juliflora extract D.

Figure 3 - Zones of inhibition observed for Staphylococcus aureus grown on MullerHinton media with Coomassie Brilliant Blue for Mouthrinse A, B, C, Ciprofloxacin CF and P. juliflora extract D.

Figure 4 - Zone of inhibition observed for Prevotella intermedia grownon WilkinsChalgren Blood Agar for Mouthrinse A, B, C, Ciprofloxacin CF and P.juliflora extract D.

5. 6.


Journal of Pharmacy Research Vol.4.Issue 7. July 2011


Hari Prasad O et al. / Journal of Pharmacy Research 2011,4(7),2149-2151

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. periodontal, 1976 47: 373. Slots J. subjingival microflora & periodontal disease. J Clin Periodontal, 1979; 6:351. Portenier I, Waltimo TMT and Haapasalo M. Enterococcus faecalis the root canal survivor and star in post treatment disease. Endod Topics, 2003; 6:135-59. Schleifer KH, Klipper-Balz R. Transfer of Streptococcal faecalis and Sterptococcus faecium tothe genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. Int J Syst Bacteriol, 1984; 34:31-4 Kayaoghu G and Orstavik D. Virulence factors of Enterococcus faecalis: Relationship to endodontic disease. Crit Rev Oral Biol Med, 2004; 15(5): 308-320. Scannapicco FA, Bush RB and Paju S: Associations between periodontal disease and risk for atherosclerosis, Cardiovascular disease, and stroke: a systemic review, Ann Periodontal, 2003; 8:38. Chlorhexidine Official FDA information, side effects and uses. Drug information online http:// (accessed on 14 March, 2011). Lisa S.Natural remedies for treating periodontal disease,2009. http: for_treating_ periodontal.html? cat=5 (Accessed on 10 March 2011). Pramod Kumar, Shahid H, Ansari and Javed Ali. Herbal Remedies for the treatment of Periodontal Disease A Patent Review. Recent Patents on Drug Delivery & Formulation, 2009; 3: 221-228. Shankarmurthy P and Siddiqui S. Antibiotic activity of leaf extracts of Prosopis juliflora.Journal of Scientific and Industrial Research, 1948; 7: 188. Ahmad A, Ali KK, Ahmad VU and Qazi S. Antibacterial activity of juliflorine isolated from Prosopis juliflora. Planta Medica, 1986; 1: 285-288. Aqeel A, Khursheed AK, Viqaruddin A and Sabiha Q. Antimicrobial activity of julifloricine isolated from Prosopis juliflora. Arzneim. - Forsch. Drug Research, 1989; 39: 652-655. Kanthasamy A, Subramanian S and Govindasamy S. Bactericidal and fungicidal effects of Prosopis juliflora alkaloidal fraction. Indian Drugs, 1989a; 26: 390-394. Ahmad A, Khursheed AK, Sabiha Q and Viqaruddin A. Antifungal activity of some hydrosoluble Prosopis juliflora alkaloids. Fitoterapia, 1989a; 60: 86-89. Kanthasamy A, William S and Govindasamy S. Hemolytic effects of Prosopis juliflora alkaloids. Current Science, 1989b; 58: 142-144. Batatinha MJM. Investigations on toxic influences of Prosopisjuliflora D.C. (Algarobeira) on cell cultures as well as on ruminal fermentation in cattle (in vitro). Dissertao (mestrado)Veterinary Medicine University, Hannover, 1997; 189. Hari Prasad O, Navya A, Sarma PVGK and Uma Maheswari Devi. Anti-microbial and antiproliferative activity of P. juliflora Leaf alkaloid extracts. 2011, (Communicated to Phytotherapy) Dr. Dukes Phytochemical and Ethnobotanical Databases. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. (Accessed on 14 March, 2011). Davidow Joie. Infusions of Healing: A Treasury of Mexican-American Herbal Remedies. Simon and Schuster Inc., 1999. 149. Desert USA. 13 June 2001. {www.desertusa/jan97/dusmesquite.html} Kay and Margarita Artschwager. Healing with Plants in the American and Mexican West. Tuscon: The University of Arizona Press, 1996. 221-224. Socransky SS, Haffajee AD, Cugini MA, Smith C and Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol, 1998; 25: 134-44. Nonnenmacher C, Stelzel M and Susin C. Periodontal microbiota in patients with coronary artery disease measured by real-time polymerase chain reaction: A case-control study. J Periodontol, 2007; 78:1724-1730. Raghavendra MP, Satish S and Raveesha KA. Alkaloid extracts of Prosopis juliflora (Sw.) DC. (Mimosaceae) against Alternaria alternate. Journal of Biopesticides, 2009; 2(1): 56-59. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. Vol. 31 No. 1. M100-S21. January 2011. Wayne, PA: Clinical and Laboratory Standards Institute. Steinberg D and Friedman M. Sustained release drug delivery devices for treatment of dental diseases; In: Tyle P, Ed. Drug delivery devices: Fundamentals and applications. New York: Marcel Dekker 1998; 491-515. Lang NP, Hotz P, Graf H, Geering AH, Saxer UP and Sturzenberger OP. Effects of supervised Chlorhexidine mouthrinses in children. J Periodont Res, 1982; 17:101-111 Lamster IB, Alfano MC, Seiger MC and Gordon JM. The effect of Listerine antiseptic on reduction of existing plaque and gingivitis. Clin Prev Dent, 1983; 5:12-16. Grossman E, Reiter G, Sturzenberger OP, De la Rosa M, Dickinson TD and Ferretti GA. Six-month study of the effects of a Chlorhexidine mouthrinse on gingivitis in adults. JPeriodont Res, 1986; 21: 33-38. Mandel ID. Chemotherapeutic agents for controlling plaque and gingivitis. J Clin Periodontol, 1988; 15:488-498. DePaola LG, Overholser CD, Meiller TF, Minah GE and Niehaus C. Chemotherapeutic inhibition of supragingival dental plaque and gingivitis development. J Clin Periodontol, 1989; 16(5):311-5. Overholser CD, Meiller TF, DePaola LG, Minah GE and Niehaus C. Comparative effects of 2 chemotherapeutic mouthrinses on the development of supragingival dental plaque and gingivitis J Clin Periodontol, 1990;17(8):575-9. Tambekar DH, Khante BS, Chandak BR, Tltare AS, Boralkar SS and Aghadte SN. Screening of Antibacterial potentials of some medicinal plants from Melghat forest in India. Afr. J. Tras. CAM, 2009; 6(3): 228-232. Newman MG. Anaerobic oral and dental infections. Rev Infect Dis, 1984; 6 Suppl 1:S107S114.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 7. July 2011