|Year : 2016 | Volume
| Issue : 4 | Page : 456-462
Minimum inhibitory concentration of the plant extracts' combinations against dental caries and plaque microorganisms: An in vitro study
BR Chandra Shekar1, Ramesh Nagarajappa2, Richa Jain3, S Suma4, Rupal Singh3, Rupesh Thakur3
1 Department of Public Health Dentistry, JSS Dental College and Hospital, Jagadguru Sri Shivarathreeshwara University, Mysore, Karnataka, India
2 Department of Public Health Dentistry, Institute of Dental Sciences, Bhubaneshwar, Odisha, India
3 Center for Scientific Research and Development, People's University, Bhopal, Madhya Pradesh, India
4 Department of Orthodontics, JSS Dental College and Hospital, Jagadguru Sri Shivarathreeshwara University, Mysore, Karnataka, India
|Date of Web Publication||15-Dec-2016|
B R Chandra Shekar
Department of Public Health Dentistry, JSS Dental College and Hospital, Jagadguru Sri Shivarathreeshwara University, JSS Medical Institutions Campus, Mysore - 570 015, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Oral health status has witnessed marked advances in many industrialized countries. However, dental caries is consistently increasing in developing countries, and periodontal diseases are among most common afflictions to humankind. Approach best suited for developing countries is to focus on the prevention with innovative strategies. Hence, evolution of novel, innovative strategies to prevent dental caries and periodontal diseases is need of hour. Objective: To determine minimum inhibitory concentration (MIC) of combinations of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, and Psidium guajava against dental caries and plaque microorganisms and to qualitatively identify various phytochemical constituents in individual plant extracts and their quadruple combinations. Materials and Methods: MIC of the combinations of A. nilotica, M. koenigii L. Sprengel, Eucalyptus hybrid, and P. guajava on Streptococcus mutans, Lactobacillus acidophilus (dental caries bacteria), Streptococcus sanguis, Streptococcus salivarius (primary plaque colonizers), Fusobacterium nucleatum (secondary plaque colonizer), and Porphyromonas gingivalis (tertiary plaque colonizer) was determined using broth dilution method. Series of dilutions of quadruple combinations ranging from 0.05% to 1.5% were prepared. 100 μL of each serial dilution of quadruple combinations was added to each tube containing bacterial culture. The optical density was noted after incubation in each tube to estimate the MIC for each bacterium. Results: MIC of the polyherbal combinations on S. mutans, S. sanguis, S. salivarius, L. acidophilus, F. nucleatum, and P. gingivalis was found to be 0.25%, 0.05%, 0.05%, 0.1%, 0.25%, and 0.25%, respectively. Conclusion: The quadruple combinations of these four plant extracts could be considered in the evolution of an indigenous polyherbal mouth rinse as the formulation inhibited all the bacteria tested in the present study at low concentrations.
Keywords: Dental caries, dental plaque, minimum inhibitory concentration, periodontal diseases, Streptococcus mutans
|How to cite this article:|
Chandra Shekar B R, Nagarajappa R, Jain R, Suma S, Singh R, Thakur R. Minimum inhibitory concentration of the plant extracts' combinations against dental caries and plaque microorganisms: An in vitro study. J Indian Assoc Public Health Dent 2016;14:456-62
|How to cite this URL:|
Chandra Shekar B R, Nagarajappa R, Jain R, Suma S, Singh R, Thakur R. Minimum inhibitory concentration of the plant extracts' combinations against dental caries and plaque microorganisms: An in vitro study. J Indian Assoc Public Health Dent [serial online] 2016 [cited 2020 Jul 5];14:456-62. Available from: http://www.jiaphd.org/text.asp?2016/14/4/456/195842
| Introduction|| |
Health promotion measures are incomplete without oral health promotion and prevention of oral diseases that have a considerable influence on general health. Oral diseases continue to present an upward trend in the developing countries despite marked improvement in oral health status in developed countries. Dental caries and periodontal diseases are major dental public health problems in many developing countries. The cost of treating dental caries and periodontal diseases is quite expensive and not a practical option for developing countries. At the same time, cost of neglect is also exigent in view of their established association with systemic health.,,, There is a pressing need for promoting indigenous preventive measures that are acceptable, cost-effective, and easily available.
Antimicrobial mouth rinses have also been recommended as adjuncts for mechanical plaque control methods. Chlorhexidine gluconate is the most commonly used antiplaque agent. However, its long-term use has been reported with altered taste sensation, staining of teeth, and development of resistant microorganisms. This necessitates the evolution of some innovative strategies that act against microorganisms involved in the causation of dental caries and periodontal diseases. One such strategy would be to explore the colossal wealth of medicinal plants richly available in natural world.
The majority of the published literature assessed the efficacy of individual plant extracts on bacteria involved in causing either dental caries or periodontal diseases. The combination of these plant extracts that can combat both dental caries and periodontal pathogens is not investigated so far. A methodical and orderly evaluation of plant extracts and their combinations presents an ideal approach in the evolution of novel drugs from plants.Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, and Psidium guajava extracts at 10% concentration have been found to inhibit the growth of dental caries and plaque bacteria in our previous in vitro studies.,, It is essential to determine the minimum inhibitory concentration (MIC) of combinations of these plant extracts on dental caries and plaque bacteria before assessing feasibility of using this herbal formulation as a mouth rinse for daily use. This will enable us to determine the concentration of mouth rinse if such attempts are made. In this background, the present study was undertaken to determine MIC of the combinations of A. nilotica, M. koenigii L. Sprengel, Eucalyptus hybrid, and P. guajava against dental caries and plaque microorganisms and to qualitatively assess the phytochemical constituents present in individual plant extracts and their quadruple combinations.
| Materials and Methods|| |
This in vitro study was carried out over a period of 6 months from April to September 2014 at the center for scientific research and development, People's University, Bhopal. The research protocol was approved by the Institutional Review Board of Pacific Academy of Higher Education and Research University, Udaipur. The branches of A. nilotica, M. koenigii L. Sprengel, Eucalyptus hybrid, and P. guajava were collected, identified, and validated by a taxonomist. Healthy leaves were cut from their branches, washed, and shade dried for 3–4 weeks at room temperature. The dried leaves were hand crushed, and the fine powder was prepared using a mixer. The powders were stored in coded airtight plastic bottles in refrigerator at 4°C. Ethanolic extracts of these plants were obtained using Soxhlet apparatus. The extraction process is diagrammatically depicted in [Figure 1].
The stock solutions of the individual plants were prepared by dissolving 100 mg of the extract in 1000 µl of dimethyl sulfoxide. The quadruple combinations of plant extracts were prepared by mixing equal quantities of the stock solutions of individual extracts.
The MIC of the combinations of A. nilotica, M. koenigii L. Sprengel, Eucalyptus hybrid, and P. guajava on Streptococcus mutans, Lactobacillus acidophilus (dental caries bacteria), Streptococcus sanguis, Streptococcus salivarius (primary plaque colonizers), Fusobacterium nucleatum (secondary plaque colonizer), and Porphyromonas gingivalis (tertiary plaque colonizer) was carried out using broth dilution method.
Culturing of microorganism, inoculum development, and MIC determination were carried out in anaerobic chamber with 5% CO2. All the four extracts were mixed in 1:1:1:1 proportion and then appropriately diluted at different serial dilutions ranging from 0.05% to 1.5%. The inoculum of cultures (all six cultures) was developed in broth medium [Figure 2].
|Figure 2: Revival of bacteria and preparation of serial dilutions of plant extracts' combinations for minimum inhibitory concentration estimation|
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The cultures were then incubated and subsequently, serially diluted to reach the density of 2 × 104 cells per ml. Cell counting was done using hemocytometer. Two milliliters of cooked meat broth was dispensed in tubes, and 100 μL of cell culture was inoculated in it. Then, 100 μL of different concentration of extract was added to each tube. Each experiment was carried out in a triplicate set. Growth control was run in parallel with every experiment. All the experimental tubes were incubated in anaerobic jars for 48 h. After completion of incubation period, the optical density was measured at 600 nm using spectrophotometer. MIC was defined as the minimum concentration of extract that caused 20% inhibition in growth of test microorganism., The percentage of bacterial inhibition by each extract was computed using the following equation:
Qualitative assay of phytochemicals
Various phytochemical constituents present in the individual plant extracts, and the quadruple combinations were assessed using the following biochemical tests.,
Detection of alkaloids
Fifty milligrams of solvent-free extract was taken in a test tube. Few drops of dilute hydrochloric acid was added drop by drop to this test tube and stirred. The filtrate was then tested with Mayer's reagent, Wagner's reagent, and Hager's reagent. The appearance of a white or creamy precipitate with Mayer's reagent indicated the presence of alkaloids. The development of a reddish brown precipitate with Wagner's reagent confirmed the presence of alkaloids. The appearance of a prominent yellow precipitate with Hager's reagent indicated the test to be positive.
Detection of anthraquinones
Fifty milligrams of the extract was dissolved in distilled water. Two milliliters of the extract was taken in a test tube. Then, 1 ml dilute ammonia solution was added to the test tube and shaken vigorously. The appearance of pink color indicated the presence of anthraquinones.
Detection of terpenoids
The extract was taken in a test tube and mixed with 2 ml of chloroform. To this, concentrated sulfuric acid (H2 SO4) (3 ml) was added to form a layer. The development of reddish brown coloration at the interface confirmed the presence of terpenoids.
Detection of saponins
Fifty milligrams of the extract was dissolved in 20 ml of distilled water. This suspension was shaken for 15 min in a graduated cylinder. The presence of saponins was confirmed by the formation of a 2 cm layer of foam.
Detection of flavonoids
Magnesium and hydrochloric acid reduction test
Fifty milligrams of the extract was dissolved in 5 ml of alcohol in a test tube. To this, few fragments of magnesium ribbon and concentrated hydrochloric acid were added drop by drop. The presence of flavonoids was confirmed by the development of pink to crimson color.
Detection of tannins
Ferric chloride test
Fifty milligrams of the extract was dissolved in 5 ml of alcohol in a test tube. To this, few drops of 5% ferric chloride were added. The appearance of dark green color established the presence of tannins.
Detection of cardiac glycosides
Fifty milligrams of the extract was dissolved in distilled water and then filtered. To 2 ml of filtrate, 1 ml of glacial acetic acid, a drop of ferric chloride, and a drop of concentrated sulfuric acid were added. The development of green-blue at the upper layer and reddish-brown at the junction of two layers confirmed the presence of cardiac glycosides.,
| Results|| |
The details of four plants assessed for their efficacy in the present study are presented in [Table 1]. The detail of dental caries and plaque bacteria that includes primary, secondary, and tertiary plaque colonizers is presented in [Table 2].
|Table 1: Plant extracts used in the present study and their yield using Soxhlet extraction process|
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|Table 2: Details of the bacteria used for antimicrobial efficacy testing|
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Minimum inhibitory concentration on dental caries and plaque bacteria
MIC of the combinations of A. nilotica, M. koenigii L. Sprengel, Eucalyptus hybrid, and P. guajava on S. mutans, S. sanguis, S. salivarius, L. acidophilus, F. nucleatum, and P. gingivalis was found to be 0.25%, 0.05%, 0.05%, 0.1%, 0.25%, and 0.25%, respectively [Table 3]a and [Table 3]b.
|Table 3a: Percentage of bacterial inhibition at different serial dilutions of quadruple combinations of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, and Psidium guajava on Streptococcus mutans, Streptococcus sanguinis, and Streptococcus salivarius|
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|Table 3b: Percentage of bacterial inhibition at different serial dilutions of quadruple combinations of Acacia nilotica, Murraya koenigii L. Sprengel, Eucalyptus hybrid, and Psidium guajava on Lactobacillus acidophilus, Fusobacterium nucleatum, and Porphyromonas gingivalis|
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Qualitative assay of phytochemical constituents
A. nilotica revealed the presence of anthraquinones, flavonoids, tannins, and cardiac glycosides. M. koenigii L. Sprengel showed the presence of tannins and cardiac glycosides. Eucalyptus hybrid revealed the presence of terpenoids, saponins, flavonoids, tannins, and cardiac glycosides. P. guajava was found to contain anthraquinones, terpenoids, flavonoids, tannins, and cardiac glycosides. Quadruple combinations of plant extracts revealed the presence of alkaloids, flavonoids, terpenoids, tannins, saponins, anthraquinones, and cardiac glycosides [Table 4].
|Table 4: Phytochemical constituents present in individual plant extracts and their quadruple combinations|
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| Discussion|| |
Herbs are making a comeback and their “renaissance” is occurring throughout the world. In today's world, the products derived from herbs signify safety. The synthetics in contrast to herbal products are considered unsafe to humans and environment. Herbal extracts have been used in dentistry for reducing inflammation as antioxidants, antimicrobials, antifungals, antivirals, and analgesics as well as antiplaque agents. The present study was an innovative attempt assessing the MIC of the combinations of four plant extracts on some dental caries and plaque bacteria. We could not precisely compare our results with previous published literature as this was the first of its kind where MIC of quadruple combinations of plant extracts was assessed on oral microbes.
The leaf extract combinations of A. nilotica (Babul), M. koenigii L. Sprengel (Curry Leaves), Eucalyptus hybrid (Eucalyptus), and P. guajava (Guava) inhibited the growth of S. mutans, S. sanguis, S. salivarius, L. acidophilus, F. nucleatum, and P. gingivalis with MIC of 0.25%, 0.05%, 0.05%, 0.1%, 0.25%, and 0.25%, respectively. The MIC of the combinations of plant extracts against these bacteria varied from 0.05% to 0.25%, and its antimicrobial efficacy was found to be good. Deshpande and Kadam  found the MIC of ethanolic extract of A. nilotica on S. mutans to be 5 mg/ml while that from petroleum ether extract was 10 mg/ml. The MIC of the combinations of plant extracts on S. mutans in our study (0.25%) was lesser compared to the results of this study. This could be attributed to the synergistic action of phytochemicals present in combination of plant extracts. An in vitro study by Dabur et al. found the MIC of methanol extracts of A. nilotica (Babul) on Staphylococcus aureus to be 75 µg/ml. Pai et al. in their in vitro study found A. nilotica to be effective against Candida albicans. All these studies demonstrated the antimicrobial potential of A. nilotica against oral microbes similar to the findings in our study. Ningappa et al. found M. koenigii to exhibit a broad spectrum of antibacterial activity against human pathogenic bacteria, comparable to commercial antibiotics.
Nagata et al. demonstrated antibacterial activity against microorganisms involved in periodontal diseases. Among tested bacteria, P. gingivalis displayed the greatest sensitivity to macrocarpals. The MIC of macrocarpals A and B was 1 µg/ml and 0.5 µg/ml for macrocarpal C. The results were comparable to our results where MIC of the combination of four plant extracts on P. gingivalis was 0.25%. These results indicate that Eucalyptus leaf extracts may be useful as potent preventive agents for periodontal disease. Hema et al. found Gauva (P. guajava) to be effective against Pseudomonas lundensis, Aspergillus niger, and Aspergillus flavus. Gonçalves et al. screened the antimicrobial effect of essential oils and methanol, hexane, and ethyl acetate extracts from Guava leaves against diarrhea-causing bacteria: S. aureus, Salmonella spp., and Escherichia coli. Novelty of this research is assessment of MIC of an effective herbal formulation on bacteria involved in the causation of dental caries and periodontal diseases. This could be considered as a preliminary assessment before using this formulation as a mouth rinse for daily use and MIC estimation facilitates in determining an effective concentration when a polyherbal mouth rinse is prepared. The use of plant extract combinations has many advantages such as increased potency attributable to synergistic action of phytochemicals, slower rate of development of resistance as they are complex mixtures which can make microbial adaptability difficult.
This in vitro study assessed MIC on a few dental caries and plaque microorganisms while assessment on other secondary and tertiary plaque colonizers could offer a more comprehensive assessment. The quantitative assessment using high-pressure liquid chromatography rather than qualitative assay could highlight the concentration of active ingredients in these plant extracts and their combinations.
| Conclusion|| |
The plant extracts' combinations containing A. nilotica, M. koenigii L. Sprengel, Eucalyptus hybrid, and P. guajava inhibited microorganisms involved in dental caries and periodontal diseases at low concentrations. Hence, these polyherbal extracts' combinations may be considered in the preparation of an indigenously developed mouth rinse for daily use. This could be a simple and effective alternate to chlorhexidine for preventing and controlling dental caries and plaque-induced dental diseases.In vivo studies assessing their antimicrobial efficacy are recommended to validate these results.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Kwan SY, Petersen PE, Pine CM, Borutta A. Health-promoting schools: An opportunity for oral health promotion. Bull World Health Organ 2005;83:677-85.
Botelho MA, Santos RA, Martins JG, Carvalho CO, Paz MC, Azenha C, et al
. Efficacy of a mouth rinse based on leaves of the neem tree (Azadirachta indica
) in the treatment of patients with chronic gingivitis: A double-blind, randomized, controlled trial. J Med Plants Res 2008;2:341-6.
Weidlich P, Cimões R, Pannuti CM, Oppermann RV. Association between periodontal diseases and systemic diseases. Braz Oral Res 2008;22 Suppl 1:32-43.
Fentoglu O, Bozkurt FY. The Bi-directional relationship between periodontal disease and hyperlipidemia. Eur J Dent 2008;2:142-6.
Agueda A, Echeverría A, Manau C. Association between periodontitis in pregnancy and preterm or low birth weight: Review of the literature. Med Oral Patol Oral Cir Bucal 2008;13:E609-15.
Mealey BL, Oates TW; American Academy of Periodontology. Diabetes mellitus and periodontal diseases. J Periodontol 2006;77:1289-303.
Eley BM. Antibacterial agents in the control of supragingival plaque – A review. Br Dent J 1999;186:286-96.
Gupta P, Nahata A, Dixit VK. An update on Murraya koenigii
Spreng: A multifunctional Ayurvedic herb. J Chin Integr Med 2011;9:824-33.
Chandrashekar BR, Nagarajappa R, Singh R, Thakur R. An in vitro
study on the anti-microbial efficacy of ten herbal extracts on primary plaque colonizers. J Young Pharm 2014;6:33-9.
Chandrashekar BR, Nagarajappa R, Singh R, Thakur R. Antimicrobial efficacy of the combinations of Acacia nilotica
, Murraya kinigii
L sprengel, Eucalyptus
hybrid and Psidium guajava
on primary plaque colonizers. J Basic Clin Pharm 2014;5:115-9.
Chandrashekar BR, Nagarajappa R, Singh R, Thakur R. Antimicrobial efficacy of Acacia nilotica
, Murraya koenigii
L. sprengel, Eucalyptus
hybrid and Psidium guajava
on primary plaque colonizers –
An in vitro
comparison between hot and cold extraction process. J Indian Soc Periodontol 2015;9:174-9.
Wiegand I, Hilpert K, Hancock RE. Agar and broth dilution methods to determine the minimum inhibitory concentration (MIC) of antibacterial substances. Nat Protoc 2008;3:163-75.
Bussmann RW, Malca-Garcia G, Sharon GD, Chati G, Diaz D, Pourmand K. Minimum inhibitory concentrations of medicinal plants used in Northern Peru as antibacterial remedies. J Ethnopharmacol 2010;132:101-8.
Singh D, Singh P, Gupta A, Solanki S, Sharma E, Nema R. Qualitative estimation of the presence of bioactive compound in Centella asiatica
: An important medicinal plant. Int J Life Sci Med Sci 2012;2:5-7.
Deshpande SN, Kadam DG. Phytochemical analysis and antibacterial activity of Acacia nilotica
against Streptococcus mutans
. Int J Pharm Pharm Sci 2013;5:236-8.
Dabur R, Gupta A, Mandal TK, Singh DD, Bajpai V, Gurav AM, et al
. Antimicrobial activity of some Indian medicinal plants. Afr J Tradit Complement Med 2007;4:313-8.
Pai MB, Prashant GM, Murlikrishna KS, Shivakumar KM, Chandu GN. Antifungal efficacy of Punica granatum, Acacia nilotica, Cuminum cyminum
and Foeniculum vulgare
on Candida albicans
: An in vitro
study. Indian J Dent Res 2010;21:334-6.
Ningappa MB, Dhananjaya BL, Dinesha R, Harsha R, Srinivas L. Potent antibacterial property of APC protein from Curry leaves (Murraya koenigii
L.). Food Chem 2010;118:747-50.
Nagata H, Inagaki Y, Yamamoto Y, Maeda K, Kataoka K, Osawa K, et al.
Inhibitory effects of macrocarpals on the biological activity of Porphyromonas gingivalis
and other periodontopathic bacteria. Oral Microbiol Immunol 2006;21:159-63.
Hema R, Kumaravel S, Elanchezhiyan N. Antimicrobial activity of some of the South-Indian spices and herbals against food pathogens. Global J Pharmacol 2009;3:38-40.
Gonçalves FA, Neto MA, Bezerra JN, Macrae A, De Sousa QV, Fonteles-Filho AA, et al
. Antibacterial activity of Guava, Psidium guajava
linnaeus, leaf extracts on diarrhea-causing enteric bacteria isolated from seabob shrimp, Xiphopenaeus kroyeri
(Heller). Rev Inst Med Trop S Paulo 2008;50:11-5.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]