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ORIGINAL ARTICLE

Minimum inhibitory concentration of the plant extracts’

combinations against dental caries and plaque microorganisms:
An in vitro study
B. R. Chandra Shekar, Ramesh Nagarajappa1, Richa Jain2, S. Suma3, Rupal Singh2, Rupesh Thakur2
Departments of Public Health Dentistry and 3Orthodontics, JSS Dental College and Hospital, Jagadguru Sri Shivarathreeshwara
University, Mysore, Karnataka, 1Department of Public Health Dentistry, Institute of Dental Sciences, Bhubaneshwar, Odisha, 2Center for
Scientific Research and Development, People’s University, Bhopal, Madhya Pradesh, India

Address for correspondence:

Dr. 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, India. E‑mail: drchandrubr@yahoo.com

ABSTRACT:
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.
Key words:
Dental caries, dental plaque, minimum inhibitory concentration, periodontal diseases, Streptococcus mutans

INTRODUCTION developing countries despite marked improvement in

Health promotion measures are incomplete without oral
health promotion and prevention of oral diseases that
have a considerable influence on general health.[1] Oral
diseases continue to present an upward trend in the
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oral health status in developed countries. Dental caries
and periodontal diseases are major dental public health
problems in many developing countries.[2] The cost of
treating dental caries and periodontal diseases is quite
This is an open access article distributed under the terms of the Creative
Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows
others to remix, tweak, and build upon the work non‑commercially, as long as the
author is credited and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com

How to cite this article: Chandra Shekar BR, Nagarajappa R, Jain R,

DOI: Suma S, Singh R, Thakur R. Minimum inhibitory concentration of the plant
10.4103/2319-5932.195842 extracts' combinations against dental caries and plaque microorganisms: An
in vitro study. J Indian Assoc Public Health Dent 2016;14:456-62.

© 2016 Journal of Indian Association of Public Health Dentistry | Published by Wolters Kluwer - Medknow 456
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Shekar, et al.: MIC of a polyherbal extract on dental caries and plaque bacteria

expensive and not a practical option for developing and the fine powder was prepared using a mixer. The
countries. At the same time, cost of neglect is also powders were stored in coded airtight plastic bottles in
exigent in view of their established association with refrigerator at 4°C. Ethanolic extracts of these plants
systemic health.[3‑6] There is a pressing need for promoting were obtained using Soxhlet apparatus. The extraction
indigenous preventive measures that are acceptable, process is diagrammatically depicted in Figure 1.
cost‑effective, and easily available.
The stock solutions of the individual plants were prepared
Antimicrobial mouth rinses have also been recommended by dissolving 100 mg of the extract in 1000 µl of dimethyl
as adjuncts for mechanical plaque control methods. sulfoxide. The quadruple combinations of plant extracts
Chlorhexidine gluconate is the most commonly used were prepared by mixing equal quantities of the stock
antiplaque agent. However, its long‑term use has been solutions of individual extracts.
reported with altered taste sensation, staining of teeth,
and development of resistant microorganisms.[7] This The MIC of the combinations of A. nilotica, M. koenigii
necessitates the evolution of some innovative strategies L. Sprengel, Eucalyptus hybrid, and P. guajava on
that act against microorganisms involved in the causation Streptococcus mutans, Lactobacillus acidophilus (dental
of dental caries and periodontal diseases. One such caries bacteria), Streptococcus sanguis, Streptococcus
strategy would be to explore the colossal wealth of salivarius  (primary plaque colonizers), Fusobacterium
medicinal plants richly available in natural world. nucleatum  (secondary plaque colonizer), and
Porphyromonas gingivalis (tertiary plaque colonizer) was
The majority of the published literature assessed the carried out using broth dilution method.
efficacy of individual plant extracts on bacteria involved
in causing either dental caries or periodontal diseases. Culturing of microorganism, inoculum development,
The combination of these plant extracts that can combat and MIC determination were carried out in anaerobic
both dental caries and periodontal pathogens is not chamber with 5% CO2. All the four extracts were mixed
investigated so far. A methodical and orderly evaluation in 1:1:1:1 proportion and then appropriately diluted at
of plant extracts and their combinations presents an different serial dilutions ranging from 0.05% to 1.5%. The
ideal approach in the evolution of novel drugs from inoculum of cultures (all six cultures) was developed in
plants.[8] Acacia nilotica, Murraya koenigii L. Sprengel, broth medium [Figure 2].
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. [9‑11] 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, Figure 1: Step‑wise procedure of Soxhlet extraction

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Shekar, et al.: MIC of a polyherbal extract on dental caries and plaque bacteria
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.[12,13] The
percentage of bacterial inhibition by each extract was
computed using the following equation:
Percentage Inhibition
Opticaldensity in control − Opticaldensity in test set
= ×100
Opticaldensity in control
Qualitative assay of phytochemicals
Various phytochemical constituents present in
the individual plant extracts, and the quadruple
combinations were assessed using the following
biochemical tests.[14,15]
Figure 2: Revival of bacteria and preparation of serial dilutions of plant
extracts’ combinations for minimum inhibitory concentration estimation
Journal of Indian Association of Public Health Dentistry
Vol. 14, Issue 4, | October-December 2016
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
Salkowski test
The extract was taken in a test tube and mixed with 2 ml
of chloroform. To this, concentrated sulfuric acid (H2SO4)
(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
Foam test
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
Keller–kiliani test
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
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Shekar, et al.: MIC of a polyherbal extract on dental caries and plaque bacteria

of green‑blue at the upper layer and reddish‑brown at the cardiac glycosides. Quadruple combinations of plant
junction of two layers confirmed the presence of cardiac extracts revealed the presence of alkaloids, flavonoids,
glycosides.[14,15] terpenoids, tannins, saponins, anthraquinones, and
cardiac glycosides [Table 4].

RESULTS
DISCUSSION
The details of four plants assessed for their efficacy in
the present study are presented in Table 1. The detail of Herbs are making a comeback and their “renaissance”
dental caries and plaque bacteria that includes primary, is occurring throughout the world. In today’s world,
secondary, and tertiary plaque colonizers is presented the products derived from herbs signify safety. The
in Table 2. synthetics in contrast to herbal products are considered
unsafe to humans and environment. Herbal extracts
Minimum inhibitory concentration on dental caries have been used in dentistry for reducing inflammation
and plaque bacteria as antioxidants, antimicrobials, antifungals, antivirals,
MIC of the combinations of A. nilotica, M. koenigii L. and analgesics as well as antiplaque agents. The present
Sprengel, Eucalyptus hybrid, and P. guajava on S. mutans, study was an innovative attempt assessing the MIC of
S. sanguis, S. salivarius, L. acidophilus, F. nucleatum, the combinations of four plant extracts on some dental
and P. gingivalis was found to be 0.25%, 0.05%, 0.05%, caries and plaque bacteria. We could not precisely
0.1%, 0.25%, and 0.25%, respectively [Tables 3a and b]. 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
Qualitative assay of phytochemical constituents microbes.
A. nilotica revealed the presence of anthraquinones,
flavonoids, tannins, and cardiac glycosides. M. koenigii
The leaf extract combinations of A. nilotica  (Babul),
L. Sprengel showed the presence of tannins and cardiac
M. koenigii L. Sprengel  (Curry Leaves), Eucalyptus
glycosides. Eucalyptus hybrid revealed the presence
hybrid  (Eucalyptus), and P. guajava  (Guava) inhibited
of terpenoids, saponins, flavonoids, tannins, and
the growth of S. mutans, S. sanguis, S. salivarius,
cardiac glycosides. P. guajava was found to contain
L. acidophilus, F. nucleatum, and P. gingivalis with MIC
anthraquinones, terpenoids, flavonoids, tannins, and
of 0.25%, 0.05%, 0.05%, 0.1%, 0.25%, and 0.25%,
respectively. The MIC of the combinations of plant extracts
Table 1: Plant extracts used in the present study and against these bacteria varied from 0.05% to 0.25%,
their yield using Soxhlet extraction process and its antimicrobial efficacy was found to be good.[16]
Plant Botanical name Family Yield (%) Deshpande and Kadam[15] found the MIC of ethanolic
Babul Acacia nilotica Leguminosae 19 extract of A. nilotica on S. mutans to be 5 mg/ml while
Curry Murraya koenigii Rutaceae 10.8 that from petroleum ether extract was 10 mg/ml. The
L. Sprengel MIC of the combinations of plant extracts on S. mutans
Eucalyptus Eucalyptus hybrid Myrtaceae 31.5 in our study (0.25%) was lesser compared to the results
(Eucalyptus of this study. This could be attributed to the synergistic
camaldulensis × action of phytochemicals present in combination of
Eucalyptus ovata)
plant extracts. An in vitro study by Dabur et al.[16] found
Guava Psidium Guajava Myrtaceae 25.1
the MIC of methanol extracts of A. nilotica  (Babul) on

Table 2: Details of the bacteria used for antimicrobial efficacy testing

Bacteria ATCC Selective media used Type of hemolysis Media for antimicrobial
number for revival on blood agar plate efficacy testing
Dental caries Streptococcus mutans 25,175 Brain heart infusion agar Gamma hemolysis Brain heart infusion
with 5% sheep blood agar
Lactobacillus acidophilus 314 Brain heart infusion agar Beta hemolysis Columbia 5% sheep
with 5% sheep blood blood agar
Primary plaque Streptococcus sanguinis 10,556 Brain heart infusion agar Alpha hemolysis Brain heart infusion
colonizers with 5% sheep blood agar
Streptococcus salivarius 13,419 Brain heart infusion agar Gamma hemolysis Brain heart infusion
with 5% sheep blood agar
Secondary Fusobacterium nucleatum 25,586 Brain heart infusion agar Beta hemolysis Columbia 5% sheep
plaque colonizer with 5% sheep blood blood agar
Tertiary plaque Porphyromonas gingivalis 33,277 Brain heart infusion agar Beta hemolysis Columbia 5% sheep
colonizer with 5% sheep blood blood agar
ATCC – American Type Culture Collection

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Shekar, et al.: MIC of a polyherbal extract on dental caries and plaque bacteria

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
Concentrations Streptococcus mutans Percentage Streptococcus sanguinis Percentage Streptococcus salivarius Percentage
(mean OD±SD) inhibition (mean OD±SD) inhibition (mean OD±SD) inhibition
0.05% 0.450±0.017 11.94 0.430±0.024 20.52 0.423±0.020 24.6
0.1% 0.412±0.018 19.37 0.397±0.019 26.62 0.391±0.029 30.3
0.25% 0.354±0.015 30.72 0.360±0.017 33.46 0.370±0.027 34.05
0.5% 0.300±0.011 41.29 0.345±0.013 36.23 0.326±0.013 41.89
0.75% 0.278±0.018 45.6 0.301±0.014 44.36 0.285±0.015 49.2
1.0% 0.251±0.026 50.88 0.253±0.022 53.23 0.239±0.011 57.4
1.25% 0.179±0.022 64.97 0.131±0.019 75.79 0.181±0.013 67.74
1.5% 0.125±0.019 75.54 0.089±0.015 83.55 0.149±0.010 73.44
Control (without 0.511±0.026 0.541±0.025 0.561±0.025
extract)
Percentage inhibition is computed using equation ODControl − OD test . Concentration inhibiting at least 20% of bacterial growth is considered MIC. MIC – Minimum
× 100
ODControl
inhibitory concentration, SD – Standard deviation, OD – Optical density

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
Concentrations Lactobacillus Percentage Fusobacterium Percentage Porphyromonas Percentage
acidophilus inhibition nucleatum inhibition gingivalis inhibition
(mean OD±SD) (mean OD±SD) (mean OD±SD)
0.05% 0.532±0.012 15.96 0.598±0.021 4.01 0.514±0.010 13.76
0.1% 0.493±0.023 22.12 0.516±0.032 17.17 0.492±0.023 17.45
0.25% 0.421±0.019 33.49 0.486±0.021 21.99 0.410±0.012 31.21
0.5% 0.396±0.016 37.44 0.437±0.013 29.86 0.372±0.024 37.58
0.75% 0.348±0.017 45.02 0.386±0.012 38.04 0.339±0.015 43.12
1.0% 0.275±0.018 56.56 0.321±0.013 48.48 0.269±0.018 54.87
1.25% 0.232±0.023 63.35 0.279±0.016 55.22 0.208±0.011 65.1
1.5% 0.189±0.015 70.14 0.241±0.022 61.32 0.198±0.017 66.78
Control 0.633±0.021 0.623±0.023 0.596 ± 0.011
(without extract)
Percentage inhibition is computed using equation OD Control − OD test . Concentration inhibiting at least 20% of bacterial growth is considered MIC. MIC – Minimum
× 100
OD Control
inhibitory concentration, SD – Standard deviation, OD – Optical density

Table 4: Phytochemical constituents present in individual plant extracts and their quadruple combinations
Phytochemical constituents and test Acacia Murraya koenigii L. Eucalyptus Psidium Quadruple
used nilotica Sprengel hybrid guajava combinations
Alkaloids (using Mayer’s reagent) − − − − +
Alkaloids  (using Dragendorff’s reagent) − − − − +
Anthraquinones (Borntrager’s test) + − − + +
Terpenoids (Salkowski’s test) − − + + +
Saponins (Froth and emulsion test) − − + − +
Flavonoids (Shinoda test) + − + + +
Flavonoids (alkaline reagent test) + − + + +
Tannins (ferric chloride test) + + + + +
Tannins (lead acetate test) + − + + +
Cardiac glycosides (Legal test) + + + + +
Cardiac glycosides (Keller-Kiliani test) + + + + +
+: Positive, −: Negative

Staphylococcus aureus to be 75 µg/ml. Pai et  al.[17] against Candida albicans. All these studies demonstrated
in their in vitro study found A. nilotica to be effective the antimicrobial potential of A. nilotica against oral

Journal of Indian Association of Public Health Dentistry 460

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Shekar, et al.: MIC of a polyherbal extract on dental caries and plaque bacteria
microbes similar to the findings in our study. Ningappa
et al.[18] found M. koenigii to exhibit a broad spectrum of
antibacterial activity against human pathogenic bacteria,
comparable to commercial antibiotics.
Nagata et  al. [19] 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.[20] found Gauva
(P. guajava) to be effective against Pseudomonas
lundensis, Aspergillus niger, and Aspergillus flavus.
Gonçalves et  al.[21] 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.[22]
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.
461
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Vol. 14, Issue 4, | October-December 2016