|Year : 2016 | Volume
| Issue : 2 | Page : 200-204
|Antimicrobial efficacy of three medicinal plants Glycyrrhiza glabra, Ficus religiosa, and Plantago major on inhibiting primary plaque colonizers and periodontal pathogens: An in vitro study
Hunny Sharma1, GY Yunus1, Ashok Kumar Mohapatra1, Reena Kulshrestha2, Rohit Agrawal1, Monika Kalra3
1 Department of Public Health Dentistry, Rungta College of Dental Science and Research, Bhilai, Chhattisgarh, India
2 Department of Microbiology, Rungta College of Dental Science and Research, Bhilai, Chhattisgarh, India
3 Department of Public Health Dentistry, Teerthanker Mahaveer Dental College and Research Centre, Moradabad, Uttar Pradesh, India
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|Date of Submission||01-Jan-2016|
|Date of Decision||23-Feb-2016|
|Date of Acceptance||21-Mar-2016|
|Date of Web Publication||30-May-2016|
| Abstract|| |
Introduction: From ancient times, plants with medicinal values are being tested and used in treatment of various infectious disease.
Aims and Objectives: The presentin vitro study was designed to assess the antimicrobial activity of three commonly available medicinal plants Glycyrrhiza glabra, Ficus religiosa, and Plantago major on inhibiting Primary plaque colonizers and periodontal pathogens.
Materials and Methods: Bark of G. glabra, Stem of F. religiosa, and husk of P. major were collected, crushed into fine powder, and dissolved in 67% ethanol. Extracts were then subjected to test antimicrobial efficacy against primary plaque colonizers and periodontal pathogens using Kirby-Bauer disc diffusion method. Mean zone of inhibition (ZOI) was measured by HI antibiotic zone scale. One-way ANOVA using Tukey's post hoc and t-test were applied for statistical analysis.
Results: G. glabra was found to have potential antibacterial activity against primary plaque colonizers and periodontal pathogens with highest mean ZOI measuring 9.2 ± 1.09 mm and 10.6 ± 0.54 mm at 24 h, respectively. F. religiosa showed antibacterial activity against primary plaque colonizers only at 48 h with mean ZOI of 2.6 ± 0.54 mm. P. major showed no antibacterial activity against any of the microorganism in this study. Tukey's post hoc test showed statistically nonsignificant difference between G. glabra and standard antibiotic (vancomycin 10 mcg) for periodontal pathogens.
Conclusion:G. glabra and F. religiosa showed antibacterial activity against primary plaque colonizers and periodontal pathogens. However, further studies should be undertaken to affirm the same and test their efficacy in different concentration and clinical utility.
Keywords: Antimicrobials, Ficus religiosa, Glycyrrhiza glabra, medicinal plants, phytochemicals, plant extracts
|How to cite this article:|
Sharma H, Yunus G Y, Mohapatra AK, Kulshrestha R, Agrawal R, Kalra M. Antimicrobial efficacy of three medicinal plants Glycyrrhiza glabra, Ficus religiosa, and Plantago major on inhibiting primary plaque colonizers and periodontal pathogens: An in vitro study. Indian J Dent Res 2016;27:200-4
|How to cite this URL:|
Sharma H, Yunus G Y, Mohapatra AK, Kulshrestha R, Agrawal R, Kalra M. Antimicrobial efficacy of three medicinal plants Glycyrrhiza glabra, Ficus religiosa, and Plantago major on inhibiting primary plaque colonizers and periodontal pathogens: An in vitro study. Indian J Dent Res [serial online] 2016 [cited 2020 Sep 23];27:200-4. Available from: http://www.ijdr.in/text.asp?2016/27/2/200/183135
The wide variety of microorganisms harbors human oral cavity, referring to as the oral microflora. It has been estimated that less than half of these microbacterial species harboring the oral cavity can be cultivated using anaerobic microbiological culture method and that there are likely 500–700 common oral species. Plaque-associated oral diseases are among the major health problems with dental caries, gingival and periodontal diseases being the most common oral infectious disease and are major contributors to tooth loss.,,,,,, It has been shown that early stages of gingivitis can be reversed by meticulous plaque removal through personal oral hygiene procedures while treatment of early periodontal diseases involves control of plaque deposits mainly by mechanical plaque control methods such as professional scaling and root planing which is aimed to achieve ideal plaque control.
The primary objectives of these procedures for treatment of early stages of gingivitis and chronic periodontitis are to halt disease progression and to resolve inflammation by reducing the plaque and bacterial products below the threshold capable of producing breakdown, thereby allowing repair of the affected region., However, neither the personal oral hygiene measure nor the professional mechanical plaque control alone reduces bacterial load completely.,
Medicinal plants are used naturally in pure active form or in traditional preparation in most of the countries for their therapeutic qualities. With diverse availability of phytochemicals, there is great potential for discovering novel bioactive compounds equally/more potent toward microorganisms causing widespread oral diseases. From times, many researchers had tried and tested various drugs and preventive procedures, along with testing of newer medicinal plants to utilize their medicinal values for prevention of gingival and early stages of periodontal disease by controlling bacterial levels in oral cavity. Glycyrrhiza glabra (Mulethi), Ficus religiosa (Peepal tree), and Plantago major (Isabgol) are few among them, which were abundantly used in ancient times as a traditional medicine, and very less is known about their exact medicinal property in today's world.,,
Considering all the available literature and potential medicinal properties, thisin vitro study was designed with the aim to test the antimicrobial efficacy of three medicinal plants G. glabra, F. religiosa, and P. major on inhibiting primary plaque colonizers and periodontal pathogens.
| Materials and Methods|| |
Collection of plant material
The medicinal parts, i.e., bark of G. glabra, stem of F. religiosa, and husk of P. major were collected from Chhattisgarh State Minor Forest Produce Federation Raipur, which runs its retail outlets under the name of Sanjeevani and was identified by a botanist.
Preparation of plant extract
The plants extracts were prepared with hot maceration procedure (digestion), in which the medicinal part of all the three medicinal plants (Bark of G. glabra, stem of F. religiosa, and husk of P. major) were collected and washed with distilled water and air dried at room temperature. After drying, the medicinal part of the three medicinal plants was grounded into a fine powder with a mechanical grinder. The preweighted powder (50 mg) of all the three medicinal plants were then macerated in 67% v/v of ethyl alcohol for 48 h to get the required extracts with medicinal values.
Preparation of antibiotic and medicinal plant extract sensitivity testing disc
The Kirby-Bauer disc diffusion technique described by Bauer et al.(1966) was used to determine the antibacterial activity of the three medicinal herb extracts. Sterile Whatman No. 1 paper was punched into 5 mm diameter disc size. The discs were placed in glass petri dishes and sterilized at 160°C for 1 h using hot air oven. Sterile discs were allowed to soak in the extracts for 48 h for proper absorption, after which they were removed and allowed to dry.
Collection of primary plaque colonizers and periodontal pathogens
Sample of primary plaque was collected using a sterile cotton swab by gentle swabbing on cervical half of the teeth avoiding contact with gingiva and transferred to sterile saline.
Moreover, sample of periodontal pathogens was collected using sterile paper points by gentle placing the paper point in the periodontal pocket of patient with active periodontal disease after careful removal of all supragingival and sublingual plaque and then transferring it to sterile saline.
Both the samples were then cultivated in nonselective media by incubating it overnight at 37°C till the required turbidity was observed in the test tubes.
Preparation of culture agar plates
About 20 mL of molten Mueller-Hinton (MH) agar was poured into sterile petri dishes (Borosil ®) of size 90 mm in a sterile lamellar airflow chamber equipped with ultraviolet light, and high-efficiency particulate air filter, wearing mouth mask and gloves to avoid contamination of culture media and then the media was allowed to set. Standardized concentrations of McFarland 0.5 (1 × 106 colony-forming units/mL). of overnight cultures of test isolates, i.e., primary plaque colonizers and periodontal pathogens were swabbed aseptically on the agar plates.
Disc diffusion method
The discs which had been impregnated with a series of plant extracts were placed on the MH agar surface. Each test plate comprises five discs. One positive control which is sterile filter paper disc soaked in distilled water for 48 h, one commercial antibiotic disc vancomycin 10 mcg, three treated discs with herbal extract. Ten such plates were prepared (5 for primary plaque colonizers and 5 for periodontal pathogens, respectively).
The standard antibiotic discs were vancomycin 10 mcg for primary plaque colonizers and periodontal pathogens.
The plates were then incubated at 37°C for 24, 48, and 72 h to check the antimicrobial efficacy with the laps of time. After the incubation, the plates were examined for inhibition zone. The inhibition zone was measured using HI Antibiotic Zone Scale ® (Hi Media Laboratories Pvt., Ltd., Mumbai India, 2012, Ref-PW096-3NO, size: 370 mm × 65 mm). The mean zone of inhibitions (ZOIs) were calculated and recorded. Same complete procedure was repeated for periodontal pathogens.
The entire procedure was performed by the investigator in the direct guidance of a senior microbiologist.
The collected data were classified and tabulated in Microsoft Office Excel IBM SPSS software vs 22 for windows (New York, USA). Since the data were continuous type, parametric tests were used for analysis. Mean and standard deviation were calculated. One-way analysis of variance test was used for multiple group comparisons, followed by Tukey's post hoc for group-wise comparisons, and P < 0.05 was considered statistically significant.
| Results|| |
The antimicrobial activities of the three medicinal plant species against primary plaque colonizers and periodontal pathogens, measured by the diameter of the ZOI using the disc agar diffusion assay, are shown in [Table 1] and [Table 2].
|Table 1: Antimicrobial activity of the extracts against primary plaque colonizers|
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|Table 2: Antimicrobial activity of the extracts against periodontal pathogens|
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The antimicrobial activity of G. glabra at 24, 48, and 72 h (ZOI = 10.6 ± 0.5 mm, 10.6 ± 0.89 mm, and 8.6 ± 2.40 mm) were comparable to that of standard antibiotic vancomycin 10 mcg (ZOI = 11.6 ± 1.14 mm, 11.6 ± 1.51 mm, and 10.0 ± 2.54 mm) for periodontal pathogens [Table 2], but the antimicrobial efficacy of G. glabra at 24, 48, and 72 h was markedly less (ZOI = 9.2 ± 1.09 mm, 8.4 ± 0.54 mm, and 8.0 ± 1.02 mm) lower than that of vancomycin 10 mcg (ZOI = 20.8 ± 0.83 mm, 20.6 ± 0.89 mm, and 20.8 ± 1.09 mm) for primary plaque colonizers [Table 1]. While F. religiosa showed feeble antimicrobial activity only at 48 h for both the pathogens (ZOI = 2.6 ± 0.54 mm and 3.6 ± 0.54 mm, respectively) and no activity at 24 and 72 h and P. major did not show any activity against primary plaque colonizers or periodontal pathogens at any of the time interval. The extracts of G. glabra and F. religiosa were the active toward both the pathogens. On the other hand, the other P. major did not show any activity against any of the pathogens at any of the time interval.
Although antimicrobial efficacy of G. glabra [Table 1] and [Table 2] was consistent throughout all the three-time intervals for primary plaque colonizers but post hoc results showed a statistically significant difference in the activity with P ≤ 0.00 at all the three-time interval. While the post hoc results for antimicrobial efficacy of G. glabra against periodontal pathogens showed a statistically nonsignificant difference (P = 0.098, 0.348, and 0.598) in the efficacy as compared to standard antibiotic vancomycin 10 mcg at 24, 48, and 72 h, respectively.
| Discussion|| |
Despite great improvements in the global oral health status, gingival and periodontal diseases still remain the major public oral health problems. The present antimicrobial strategies used to treat gingival and periodontal diseases consist primarily of mechanical removal of dental plaque and calculus or generalized killing of oral bacteria with antibacterial compounds. Although the use of antibacterial compounds has high efficacy in killing these oral bacteria which causes gingival and periodontal diseases but they also impose a high financial burden to individual along with many side effects.
Herbal medicines have been used for many years, and their history can be rooted from ancient civilization where their role as a primary source of medicine has been evident. Herbal medicine is still the mainstay of about 75–80% of the whole population, mainly in developing countries for primary health care because of better cultural acceptability, better compatibility with human body, and fewer side effects., Hence, efforts are being carried out to discover new alternate plant-based bioactive compounds which will be safer, easily available, and substitute standard pharmaceutical remedies.
History of herbal medicines use can be rooted from ancient civilization where their role as a primary source of medicine has been evident. G. glabra, F. religiosa, and P. major are one among them which were used in ancient times but rarely used nowadays. Although several ancient literatures are available regarding their medicinal properties yet conclusive results are still lacking.
Since this study does not utilize the aqueous extraction method. Both alcoholic and aqueous extraction method should be tested to achieve the best way to extract medicinal compounds from individual plants. Minimum inhibitory concentration should be determined for each plant separately to get maximum benefits with minimum side effects. Further,in vivo studies and clinical trials are needed before considering these medicinal plants for preparation of commercial marketed products and clinical utility. Researchers should be encouraged to conduct controlled studies to prove the effectiveness and safety of natural dental products. Such studies will provide dentists with sufficient clinical evidence before prescribing the promoted natural products for their patients. The low toxicity and low costs of these herbs should encourage further investigations leading to a better understanding of traditional herbal medicine for prevention of plaque formation and strengthening of the gums, as well as in reducing the incidence of dental or periodontal infections.
In this study, 67% ethanolic extract of G. glabra showed antimicrobial activity toward periodontal pathogens comparable to standard antibiotic vancomycin 10 mcg and good efficacy toward primary plaque colonizers. Although very promising results were observed against primary plaque colonizers and periodontal pathogens, but the potency shown was not for longer duration as the ZOI reduced from 24 to 72 h, this may have been due to overcoming of the antimicrobial effects of herbal plant extracts by the bacteria present over longer duration, or it may have also due to degeneration of phytochemicals by toxins released by bacteria over period of time. However, the same was not observed in case of standard antibiotic vancomycin 10 mcg.
Similarly, in this study, ethanolic extract of F. religiosa showed feeble antimicrobial activity toward primary plaque colonizers and periodontal pathogens only at 48 h, whereas P. major showed no antimicrobial activity against any of the microorganisms at any of the time interval.
This feeble antimicrobial activity of F. religiosa and absence of antimicrobial activity of P. major may be attributed to that fact that some of the medicinal plants do not yield their medicinal compounds to ethanolic extraction. The initial screening of plants for possible antimicrobial activities typically begin using crude aqueous or alcoholic extraction method followed by various organic extraction methods further in a later stage, and nearly all of the identifiable components from plants active against microorganism are aromatic or saturated organic compounds which are mostly obtained through initial ethanolic or methanolic extraction. Keeping this in mind, this study was designed using ethanolic extraction method for preparation of plant extracts. And since no aqueous extraction method was employed to extract medicinal compound of these medicinal plants, this would have led to such results. Furthermore, there was no measure of determination of minimum inhibitory concentration at which the medicinal plant start showing their antibacterial properties, so these limitations may have led to these feeble antimicrobial activities of F. religiosa and absence of antimicrobial activity in P. major in our present study.
This shows that although herbal plants showed very promising results still their properties are not for longer duration. This should be kept in mind while preparation and application of products prepared from these plants. Because the products prepared from these plants will required repeated application to get an effect same as that of commercially available antibiotics.
| Conclusion|| |
Dentistry is varying with induction of modern science to practice dentistry. New economical treatment modalities with fewer side effects are being developed to treat gingival and periodontal disease at early stage. Similar efforts were made in this study to discover new novel bioactive compounds which can be included in routine personal oral hygiene measure to halt these disease at early stage or prevent their development. To the authors best knowledge, the study is first of its kind because for the 1st time three new local medicinal plants are used to assess their antimicrobial activity against primary plaque colonizers and periodontal pathogens. Since there are no studies to which the results are to be compared with, and hence it is a need to test the extracts of these medicinal plants at various concentration levels by both aqueous and alcoholic extraction process for further consideration of these medicinal plants in clinical aspect.
We would like to acknowledge Dr. Ram Tiwari, Dr. Swati Verma, Dr. Anubhuti Jain, Dr. Amit Reche, Dr. Rishee Dubey, and Dr. Heena Sahni, Department of Public Health Dentistry for their excellent moral support, without them this research would have not come to light.
Financial support and sponsorship
Rungta College of Dental Science and Research, Bhilai, Chhattisgarh.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Saini R. A prospective experimental comparative study on the clinical effects of calculus dissolution based oral rinse in gingivitis patient. Int J Exp Dent Sci 2015;4:33-9.
Petersen PE, Bourgeois D, Ogawa H, Estupinan-Day S, Ndiaye C. The global burden of oral diseases and risks to oral health. Bull World Health Organ 2005;83:661-9.
Palombo EA. Traditional medicinal plant extracts and natural products with activity against oral bacteria: Potential application in the prevention and treatment of oral diseases. Evid Based Complement Alternat Med 2011;2011:680354.
Monetti M, Usin MM, Tabares S, Gonzalez A, Cabral HR, Sembaj A. The presence of periodontopathogens associated with the tumour necrosis factor-alpha expression in patients with different periodontal status. Acta Odontol Latinoam 2012;25:82-8.
Grewe JM, Gorlin RJ, Meskin LH. Human tooth mortality: A clinical-statistical study. J Am Dent Assoc 1966;72:106-12.
Caldas AF Jr. Reasons for tooth extraction in a Brazilian population. Int Dent J 2000;50:267-73.
Lundqvist C. Tooth mortality in Sweden. A statistical survey of tooth loss in the Swedish population. Acta Odontol Scand 1967;25:298-322.
Kadam A, Prasad BS, Bagadia D, Hiremath VR. Effect of Ayurvedic herbs on control of plaque and gingivitis: A randomized controlled trial. Ayu 2011;32:532-5.
O'Leary T. Proceedings from the state of the art workshop on surgical therapy for periodontitis. J Periodontol 1982;53:475-501.
Research, Science and Therapy Committee of the American Academy of Periodontology. Treatment of plaque-induced gingivitis, chronic periodontitis, and other clinical conditions. J Periodontol 2001;72:1790-800.
Newman MG, Takei HH, Klokkevold PR, Carranza FA. Carranza's Clinical Periodontology. 11th
ed. New York, US: Elseiver; 2011.
Yadav R, Yadav SK. Dental disease and its cure: A review. Asian J Pharm Clin Res 2013;6 Suppl 2:16-20.
Irani M, Sarmadi M, Bernard F, Ebrahimi Pour GH, Shaker Bazarnov H. Leaves antimicrobial activity of Glycyrrhiza glabra
L. Iran J Pharm Res 2010;9:425-8.
Singh S, Jaiswal S. Therapeutic properties of Ficus religiosa
. Int J Eng Res Gen Sci 2014;2:149-58.
Zubair M. Genetic and Environmental Effects on Polyphenols in Plantago major
. Introductory Paper at the Faculty of Landscape Planning, Horticulture and Agricultural Science 2010:1 Swedish University of Agricultural Sciences Balsgård; October, 2010.
Handa SS, Singh Khanuja SP, Longo G, Rakesh DD. Extraction Technologies for Medicinal and Aromatic Plants. Trieste: International Centre for Science and High Technology; 2008. p. 22.
Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6.
Gatto MR, Montevecchi M, Paolucci M, Landini MP, Checchi L. Prevalence of six periodontal pathogens in subgingival samples of Italian patients with chronic periodontitis. New Microbiol 2014;37:517-24.
McFarland J. Nephelometer: An instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. J Am Med Assoc 1907;14:1176-8.
Waiganjo N, Ochanda H, Yole D. Phytochemical analysis of the selected five plant extracts. Chem Mater Res 2013;3:12-7.
Kamboj VP. Herbal medicine. Curr Sci 2000;78:35-9.
Saini R. Ozone therapy in dentistry: A strategic review. J Nat Sci Biol Med 2011;2:151-3.
Department of Public Health Dentistry, Rungta College of Dental Science and Research, Bhilai, Chhattisgarh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]
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