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Bactericidal activity of ayurvedic formulation against cariogenic microorganisms
Accepted Manuscript Bactericidal activity of ayurvedic formulation against cariogenic microorganisms U. Sneha, Ramalingam Karthikeyan PII:
S1878-8181(18)30674-1
DOI:
https://doi.org/10.1016/j.bcab.2019.101026
Article Number: 101026 Reference:
BCAB 101026
To appear in:
Biocatalysis and Agricultural Biotechnology
Received Date: 6 October 2018 Revised Date:
21 November 2018
Accepted Date: 19 December 2018
Please cite this article as: Sneha, U., Karthikeyan, R., Bactericidal activity of ayurvedic formulation against cariogenic microorganisms, Biocatalysis and Agricultural Biotechnology (2019), doi: https:// doi.org/10.1016/j.bcab.2019.101026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Bactericidal activity of ayurvedic formulation against cariogenic microorganisms
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Authors Names: Sneha U and Karthikeyan Ramalingam
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Affiliations: School of Life Sciences, B.S. Abdur Rahman Crescent Institute of Science and
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Technology, Chennai, India
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1. Sneha U
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Research scholar
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School of Life Sciences, B.S. Abdur Rahman Crescent Institute of Science and Technology
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GST Road, Vandalur, Chennai -600 048, India
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Phone: +91 44 22759223; +91 44 22751347; +919940323240
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Corresponding Author Information:
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2. Dr. Karthikeyan Ramalingam
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Associate Professor
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School of Life Sciences, B.S. Abdur Rahman Crescent Institute of Science and Technology
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GST Road, Vandalur, Chennai -600 048, India
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Phone: +91 44 22759223; +91 44 22751347; Mobile +917502534668
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Email: [email protected]
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Bactericidal activity of ayurvedic formulation against cariogenic microorganisms Abstract Dental caries is an infectious disease characterized by formation of biofilm on the teeth.
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It is a major health concern worldwide and can be prevented.The dried powders of Acacia
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arabica, Terminalia chebula, Terminalia bellerica and Emblica officinalishave the potential to
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cure oral diseases and these four plant powders have been used in traditional tooth powder
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formulas in India for more than 100 years. The objective of the present study was to investigate
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the antimicrobial effect of an aqueous extract of these four plant materials against the cariogenic
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microorganisms. The Minimum Inhibitory Concentration, Minimum Bactericidal Concentration,
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kinetics of killing, and adherence assay of the aqueous plant extracts against the cariogenic
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microorganisms were determined. The results showed that the combined decoction of plant
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extracts had a high bactericidal activity against all the biofilm forming cariogenic
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microorganisms tested. Toxicity studies and infection assay on Bombyx mori proved that the
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plant decoctions were not toxic. These assays suggest that the combined decoction could act as a
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potential mouthwash effective against dental caries.
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Keywords: Cariogenic microorganisms; Time-kinetics; adherence assay; Triphala; Acacia
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Arabica; toxicity studies.
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1. Introduction
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Dental caries is a worldwide problem affecting millions of people. It is a microbial infectious
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disease caused due to the demineralization and remineralization of the tooth surface. The
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microbes convert sucrose to acids which destroy the tooth enamel by decalcification(Badria and
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Zidan, 2004). The chief bacteria which cause tooth decay are Streptococcus mutans,
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Lactobacillus casei and Actinomyces viscosus(Jenkinson and Lamont, 2005).
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Mouthwash is a potential inhibitor of dental caries and it containstriclosan, cetylpyridinium
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chloride, zinc citrate or chlorohexidine. Though they are effective, they have certain side effects
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like staining of teethand development of antimicrobial strains(Ramalingam and Amaechi, 2018).
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A number of plants like orange tree, lime tree, neem plants have been used for years as
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chewing sticks.Literature has shown that neem has antimicrobial and therapeutic effects
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suggesting its potential to be used as an endodontic irrigant(Biswas et al., 2002). Murray et 2
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al.(2008)also suggested that Morindacitrifolia juice can be formulated for use as an intracanal
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irrigant.Traditional plants like Acacia arabica (bark), Terminalia chebula(fruits), Terminalia
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bellerica(fruits) and Emblica officinalis (fruits) have the potential to treat dental caries and have
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been used traditionally in India for more than 100 years(Chouhan, 2008). A combination of T.
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chebula, T.bellericaand E. officinalis, has a number of potential uses which include free radical
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scavenging, antioxidant, anti-inflammatory, dental caries prevention, immunomodulating,
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appetite stimulation, gastric hyperacidity reduction, chemoprotective,antipyretic, antistress,
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adaptogenic,
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hepatoprotective,
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chemopreventive effects (Baliga et al., 2012; Shetty et al., 2014).The advantages of using herbal
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alternatives are cost-effectiveness,easy availability, low toxicity,increased shelf lifeand lack of
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microbial resistance (Abascal and Yarnell, 2002; Balakrishnan et al., 2018).
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anticariogenic,
anticandidal,
hypoglycemic,
antimutagenic, anticancer,
wound
healing,
radioprotective,
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antibacterial,
and
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analgesic,
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The objective of the present study was to investigate the antimicrobial effect of a mouth wash prepared from Triphala in combination with Acacia arabica.
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2. Materials and Methods
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2.1.Materials
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The dried powders Acacia arabica (bark), Terminalia chebula(fruits), Terminalia
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bellerica(fruits) and Emblica officinalis (fruits)has been used in traditional tooth powder
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formulation in India for more than 100 years and were purchased from the local traditional stores
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in Chennai. The powders were of pharmacopoeia standards with reference to “The Ayurvedic
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Pharmacopoeia of India”. These powders were weighed and prepared into individual decoctions
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as well as in mixed ratio of 7:1:1:1 (A. arabica (70%), T. chebula(10%), T. bellerica(10%), E.
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officinalis (10%)) (Karthikeyan and Amaechi, 2018).The three biofilm forming cariogenic
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bacteria used in this study Streptococcusmutans(ATCC 25175),Lactobacillus casei(ATCC 393)
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and Actinomyces viscosus(ATCC 15987) were purchased from the American Type Culture
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Collection (ATCC).The media used for the growth of these bacteria were Brain Heart Infusion
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(BHI) agar and BHI broth purchased from Himedia.
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2.2.Preparation of Decoction
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Herbal decoction was prepared as per the protocol given in Sarangdhar samhita (Tripathi.,
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2001). All the individual powders as well as the combined powders were mixed with distilled 3
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water and boiled for 5 mins on an induction stove till a strong decoction was formed. The overall
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volume reduced to 1/8th of its original volume.After the decoction was prepared, it was cooled to
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room temperature. The decoctions were then filtered and centrifuged at 4000 rpm for 10 minutes
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to get a clear solution. The supernatant was stored in falcon tubes for further analysis.
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2.3.Fourier Transform Infrared Spectroscopy (FTIR) analysis
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FTIR analysis is an important technique used to detect the different bonds present in a
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compound. The decoctions were dried and the powder was subjected to FTIR analysis in a Jasco
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FTIR 6300 spectrometerwith a scan range from 400 to 4000 cm-1having a resolution of 4cm-1to
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detect different functional groups present in the sample. The different peaks of the various
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decoction samples were observed and noted.
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2.4.Antibacterial activity
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The antibacterial activity of all the individual and combined decoctions was assayed against
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S. mutans, L. casei and A. viscosus (107 CFU/ml) using the agar well diffusion method. The
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Brain Heart Infusion (BHI) agar plates were spread with 100 µl of the culture and a well was cut
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using the cork borer. One hundred microliters of the decoctions were added intothe well and the
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plates were incubated overnight at 37°C.The assay was conducted in triplicate.
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2.5.MIC and MBC
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Plant extract was serially diluted with S. mutans cell suspension (107 CFU/ml) and was
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incubated overnight at 37ºC. The lowest concentration showing no growth was recorded as the
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minimum inhibitory concentration (MIC). For Minimum Bactericidal Concentration (MBC)
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testing, aliquots were plated onto Brain Heart Infusion (BHI) agar and incubated overnight at
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37°C. The lowest concentration showing 99.9% killing was recorded as the MBC.
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Chlorohexidine was used as positive control.MIC and MBC assays were conducted for L. casei
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and A. viscosusas well.The assays were conducted in triplicate(Karthikeyan et al., 2011;
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Ramalingam et al., 2012).
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2.6.Time kinetics
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Overnight cultures of S. mutans, L. casei, A. viscosusand mixedcultures of the three strains
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were added to plant decoctions inmicrotiter plates. Same concentrations of chlorohexidine were
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used as positive control. Following addition of the bacterial culture, 100 µl sample was retrieved
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from each plate at 1, 5, 15, 30 and 60 min and serially diluted (10-1– 10-4). The bacterial count
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was estimated using plate count method for all the dilutions(Karthikeyan et al., 2011; 4
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Karthikeyan and Amaechi, 2018).The dilution which showed countable number of microbes was
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noted along with the count of the bacteria.The experiment was conducted in triplicate.
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2.7.Adherence assay Overnight bacterial cultures containing 107 CFU/ml of S. mutans, L. casei, A. viscosusand
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mixed cultures of the three strains were added to BHI broth containing plant decoctions
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individually and in combination in the ratio 1:1. The tubes were incubated aerobically(to
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simulate conditions in the mouth) at 37°C for 24h at an angle of 30°(Ramalingam et al., 2012;
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Karthikeyan and Amaechi, 2018). Chlorohexidine 0.2% (v/v) was used as positive control. The
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contents of the tubes were poured out and the tubes were washed with distilled water. One
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milliliter of autoclaved distilled water was then added to all the tubes and the tubes were
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vortexed for a minute. Subsequently dilutions were made from this starting from 10-1 upto 10-4
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for all the samples. The bacterial count was estimated at each dilution for all the samples by plate
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count technique. The dilution which showed countable number of microbes was noted along with
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the count of the bacteria.All experiments were carried out in triplicate.
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2.8.Bombyx mori Toxicity Infection Assay
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As an invertebrate model, silkworm model is characterized by its convenience, low cost, no
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ethical issues. The presence of conserved immune response and similar pharmacokinetics
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compared to mammals make silkworm infection model suitable to examine the therapeutic
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effectiveness of antimicrobial agents (Panthee et al., 2017).Bombyx mori collected from
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sericulture units in Alangayam, Tamil Nadu was acclimatized in the lab and fed with mulberry
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leaves.
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For control studies,Bombyx mori worms (5 per set) were injected with 200 µL of the plant
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extract diluted 50% with ultrapure water. Two hundred microliter of chlorohexidine was used as
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positive control and as a negative control 200 µL of sterile distilled water was injected into the
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worms. They were incubated for a day at room temperature. The number of worms surviving
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were noted after 24 hours. The experiment was conducted in triplicate (Zhang et al., 2015; Usui
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et al., 2016).
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For carrying out the infection assay, the worms were first injected with 1 unit of bacterial
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culture S. mutans. After 15 mins, they were injected with 2 units of the plant decoctions
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individually or in combination. For the positive control, 2 units of chlorohexidine was injected
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and for negative control, 2 units of sterile distilled water was injected into the worms. They were 5
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incubated for a day at room temperature. The number of worms surviving were noted after 24
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hours.This assay was repeated with L. casei, A. viscosus and combination of all 3 bacteria. The
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experiment was conducted in triplicate (Zhang et al., 2015; Usui et al., 2016).
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All the data was statisticallyanalyzed using one-way ANOVA. Criterion for statistical
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significance was defined as P < 0.05.
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3. Results and Discussion
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3.1. Preparation of Decoction
Acacia arabica, Terminalia chebula, Terminalia bellericaand Emblica officinalis decoctions
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were prepared individually (Welihinda et al. 2006). A combined decoction ofAcacia arabica,
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Terminalia chebula, Terminalia bellericaand Emblica officinalis was preparedin the ratio of
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7:1:1:1 respectively(Karthikeyan and Amaechi, 2018).
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3.2.Fourier Transform Infrared Spectroscopy (FTIR) analysis
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Figure 1 represents the various FTIR spectra of the different decoctions.FTIR spectrum of
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decoction of Emblica officinalis showed a major peak at 3218.61 cm-1 which indicates the
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presence of phenols and alcohols. The other peaks at 1696.12 cm-1, 1691.96 cm-1, indicates the
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presence of carboxylic acids, peak at 1402.96 cm-1 indicates alkanes, peaks at 1216.86 cm-1,
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1032.69 cm-1 represent esters or ethers, the other peaks at 867.81 cm-1, 800.314 cm-1, 621.931
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cm-1, 617.793 cm-1 represent alkyl halides.FTIR spectrum of decoction of Terminalia chebula
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showed a major peak at 3200.29 cm-1 which indicates the presence of phenols and alcohols. The
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other peaks at 1696.09 cm-1, 1598.7 cm-1, indicates the presence of carboxylic acids, peak at
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1328.71 cm-1 indicates alkanes, peaks at 1193.72 cm-1, 1031.73 cm-1 and 632.637 cm-1 represent
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alkyl halides.FTIR spectrum of decoction of Terminalia belliricashowed a major peak at 3231.16
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cm-1 which indicates the presence of phenols and alcohols. The other peaks at 1696.09 cm-1,
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1602.66 cm-1 indicate the presence of carboxylic acids, peak at 1327.76 cm-1 indicates alkanes,
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peaks at 1186.04 cm-1, 1029.8 cm-1 and 417.513 cm-1 represent alkyl halides, and peak at 698.105
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cm-1 represents alkynes.FTIR spectrum of decoction of Acacia arabica showed a major peak at
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3219.68 cm-1 which indicates the presence of phenols and alcohols. The other peaks at 1606.45
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cm-1, 1442.49 cm-1 indicate the presence of carboxylic acids, peaks at 1321 cm-1, 1209.16 cm-1,
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1030.77 cm-1 and 768.494 cm-1 represent alkyl halides.FTIR spectrum of decoction of mixed
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decoction of all 4 plants showed a major peak at 3210.9 cm-1 which indicates the presence of
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phenols and alcohols. The peak at 1697.05 cm-1 indicates the presence of aldehydes, peak at
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1599.66 cm-1 represents amides, peaks at 1334.6 cm-1, 1199.61 cm-1, 1031.73 cm-1 and 616.145
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cm-1 represent alkyl halides. FTIR analysis of the Triphala showed the presence of different functional groups ranging
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from O-H alcohol (3200-3600 cm-1), C-H alkane (2850-3000 cm-1), =C-H aldehyde (2820-2850
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cm-1), acyclic ketone (1705-1725 cm-1), N-H amide (1550-1640 bending), C=C aromatic (1400-
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1600 cm-1), C-N amine (1080-1360 cm-1), C-F alkyl Halide (1000-1400 cm-1), =C-H alkene
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(675-1000 cm-1) as functional groups(Amala and Jeyaraj, 2014).
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3.3.Antibacterial activity
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The antimicrobial activity of plant decoctionswas checked against Streptococcus mutans,
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Lactobacillus caseiand Actinomyces viscosus. It wasquantitatively assessed by measuring the
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inhibition zone. Plant extract was effective with the inhibition zones ranging from 13 to 24 mm.
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The mixed decoction had a maximal zone of inhibition of 20 mm, 24 mm and 19 mm when
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compared to chlorohexidine which showed a zone of inhibition of 18 mm, 21 mm and 19 mm
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against Streptococcus mutans, Lactobacillus caseiand Actinomyces viscosusrespectively (p <
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0.05). Hence, combined decoction is a better antibacterial agent in comparison to chlorohexidine.
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Figure 2and 3 represent the photograph and graph of the zone of inhibition of the different
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microbes against the different decoctions.
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Invitro antimicrobial activity of Triphala (aqueous extract)with Staphylococcus aureus,
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Pseudomonas aeruginosa and Klebsiella pneumoniae has been reported to have inhibitory zone
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at 14mm, 10.75mm, 10mm(Amala and Jeyaraj, 2014).Gupta et al.(2014) has reported that the
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combined aqueous extract of Terminalia chebula, Emblica officinalis and Terminalia bellerica
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showed higher zone of inhibition compared to 0.2% chlorohexidine.Thomas et al. (2011) found
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the mean inhibitionzone for the aqueous extract of Triphala(combination of Emblica officinalis,
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Terminalia chebula and Terminalia bellerica)at 50%, 25%and 12.5% against S. mutans(MTCC
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strain) to be 30 mm, 28 mm, and24 mm respectively. The mean inhibition zone for the
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aqueousextract of Triphala at 50%, 25% and 12.5% against clinicalisolates of S. mutanswas
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found to be 34 mm, 30 mm, and28 mm, respectively.Prajapathi and Raol (2014) found the
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aqueous extract ofTriphala to inhibit S. mutans. The mean inhibition zone was17 mm against
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MTCC strains and 19 mm against clinicalisolates.
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3.4.MIC and MBC
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The MIC assay was performed for all the three bacteria. All the three bacteria showed
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inhibition upto the 3rd well (p < 0.05). So upto 3 fold dilution there was inhibition. Hence the
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MIC was at 1.25 mg/ml.
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MBC was performed from 1-6 wells for all the samples along with negative control and media
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control. The concentration up to which there was 99.9% reduction in bacterial load was
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considered to be as MBC. The concentration of MBC was 2.5 mg/ml(p < 0.05).
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Triphala showed minimal inhibitory concentration and minimal bactericidal concentration at
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6.25% for MTCC strain of S.mutans and 3.12% for clinical isolate of S.mutans(Thomas et al.,
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2010).MICs and MBCs of the colloidal solutions containing ZnO, CuO, TiO2 and Ag
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nanoparticles were comparable to each other and all were significantly lower than that of the
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chlorhexidine mouthrinse against both S. mutans and S. sangius(Ahrari et al., 2015).
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3.5.Kinetics Study
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Kinetics study at time points 1, 5, 15, 30 and 60 minutes was performed for all the bacteria-
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individually and in combination. Throughout the time period, the number of colonies in the
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decoction treated samples was much lesser when compared to the negative control(p < 0.05). The
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number of bacterial colonies increased rapidly from 1 minute to 60 minutes in the negative
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control. In all the treated samples, the number of microbes was gradually increased from 1
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minute to 15 minutes followed by gradual reduction(p < 0.05). The positive control did not
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contain any bacterial colonies in all the cases.Figures 4-7represent the graphs of the
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Streptococcus mutans, Lactobacillus casei, Actinomyces viscosus and combination of all three
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bacterial cultures after treatment with various decoctions.The combined decoction
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Nanoemulsions have been found to have bactericidal properties against Grampositiveand
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enteric pathogen species in various earlier reports(Teixeira et al., 2007; Hamouda et al., 1999,
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2001).Greater killing (49–94.73%) of S. mutans was observed with mixed herbal powder extract
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containing methanolic extract of triphala and Acacia arabica at 60 min when compared with
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chlorohexidine (36.56–65.93%) during kinetics study (Karthikeyan and Amaechi, 2018).
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3.6.Adherence Assay
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Adherence assay was performed with all the 3 bacteria bothindividually and as a consortium-
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Streptococcus mutans, Lactobacillus casei and Actinomyces viscosus. The number of bacterial
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colonies of all dilutions was noted the next day. In all the 4 cases, the number of bacterial
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colonies when treated with the various decoctions showed much lesser bacterial count when 8
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compared to the negative control which was untreated(p < 0.05). The number of bacterial
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colonies after being treated with the mixed decoction was 1000-fold lesser than the negative
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control bacterial count in all the cases. The positive control showed absence of bacteria in all the
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cases(p < 0.05). Figure 8 represents the graphs of bacterial count of various treated samples. Glass adherence assay used in the present study is an effective model for assessing sucrose
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dependent bacterial adhesion (Nostro et al., 2004).More inhibition of S. mutans adherenceby
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mixed herbal powder extractcontaining methanolic extract of triphala and Acacia Arabica (up to
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85.64%) at 100 µg/ml concentration has been reported when compared tochlorohexidinewhich
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showed inhibition up to 73.21% (Karthikeyan and Amaechi, 2018).
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3.7.Bombyx mori infection assay
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This study is the first study in which plant decoctions have been tested against cariogenic
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microbes in vivo using silk worm as a model organism. In control studies, the photographs of the
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worms after 24 hours of treatment are shown in figure 9. Figure 10 represents the graph
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indicating the percentage of surviving population of B. mori worms after 24 hours of
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administration of the 50% diluted decoctions and chlorohexidine.
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In the control, the worms given only plant decoctions survived 100%(p < 0.05). The worms
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injected with distilled water also survived 100%. The worms injected with chlorohexidine
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survived only 60%(p < 0.05). This proved that the plant decoctions were not toxic whereas the
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chlorohexidine was toxic.Side effects of chlorohexidine like altered taste perception, metallic
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taste, and staining of teeth have been reported earlier (Flotra et al., 1971).Chlorohexidine shows
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cytotoxicity and even causes dysplasia of the oral mucosa (Sonis et al., 1978; Cabral et al., 2007;
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De Souza et al., 2017).
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In S. mutans infection assay, worms treated with Terminalia bellerica and mixed
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decoction showed higher survival rates (65%) compared to untreated control as well as worms
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treated with chlorohexidine- both of which showed only 45% survival(p < 0.05). This showed
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that treatment with mixed decoction was better than chlorohexidine.In all the other bacterial (L.
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casei, A. viscosus and mixed culture of all 3 bacteria) infections, the plant decoctions showed
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better survival rate than the positive control(p < 0.05).Saxena et al., (2017) had reported that
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the mean colony forming units of S. mutans with Triphala was significantly reduced at 5 minutes
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when tested on human trials. The ability of these rinses to reduce plaque, freshen breath, to
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prevent or control tooth decay, to reduce gingivitis, to reduce the speed that tartar forms on the
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teeth, or to produce a combination of these effects (Kornman, 1986).
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4. Conclusion Thus the aqueous plant extract prepared from the plants- Acacia arabica, Terminalia
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chebula, Terminalia bellerica and Emblica officinalis was found to be effective against the
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common dental caries causing bacteria- Streptococcus mutans, Lactobacillus casei and
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Actinomyces viscosus.In this study, antibacterial activity, MIC, MBC, kinetics study, adherence
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assay and toxicity studies was performed. Commercially available chlorohexidine seemed to be
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an effective antibacterial agent but wascytotoxicand led to reduced survival of B. mori. The
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combined plant decoction gave 99.9% reduction in the total amount of bacteria while proving to
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be completely non-toxic to B. mori.
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5. Funding
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This work was supported by The Ministry of Ayurveda, Yoga and Naturopathy, Unani, Siddha
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and Homoeopathy (AYUSH)-EMR project(Sanction order No. Z.28015/209/2015-HPC) and
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DST Science and Engineering Research Board (SERB) [Grant number- SERB/LS-267/2014].
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effects of different concentrations of chlorhexidine. Am J Dent;20(6), 400-4. 11. Flötra, L., Gjermo, P., Rölla, G., Waerhaug, J., 1971. Side effects of chlorhexidine mouth washes. Eur. J. Oral Sci. 79, 119-25.
12. Gupta, R., Chandrashekar, B.R., Goel, P., Saxena, V., Hongal, S., Jain, M., Ganavadiya, R.,
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2014. Antimicrobial efficacy of aqueous and ethanolic extracts of Triphala on primary plaque
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colonizers: An in vitro study, Journal of Young Pharmacists. 6(3),7-13.
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13. Hamouda, T., Hayes, M.M., Cao, Z., 1999. A novel surfactant nanoemulsion with broadspectrum sporicidal activity against Bacillus spores. J. Infect. Dis.180, 1939–1949. 14. Hamouda, T., Myc, A., Donovan, B., Shih, A.Y., Reuter, J.D., Baker, J.R.,2001. A novel
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surfactant nanoemulsion with a unique non-irritant topical antimicrobial activity against
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bacteria envelopedviruses and fungi. Microbiol. Res. 156, 1–7.
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15. Jenkinson, H., Lamont, R.J., 2005. Oral microbial communities in sickness and in health.
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Trends Microbiol.13, 589-95. 16. Karthikeyan, R., Amaechi, B.T., Rawls, H.R., Lee, V.A.,2011. Antimicrobial activity of nanoemulsion on cariogenic Streptococcus mutans. Arch Oral Biol. 56, 437-445. 17. Kornman, K.S., 1986. The role of supragingival plaque in the prevention and treatment of periodontal diseases. A review of current concepts. J Periodontal Res.21(suppl), 522. 18. Murray, P.E., Farber, R.M., Namerow, K.N., Kuttler, S., Garcia-Godoy, F., 2008. Evaluation of Morindacitrifolia as an endodontic irrigant. J.Endod. 34, 66–70.
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19. Nostro, A., Cannatelli, M.A., Crisafi, A.D., Musolino, A.D., Procopio, F., Alonzo, V., 2004. Modifications
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of Streptococcus mutans by Helichrysumitalicum extract. Lett. Appl. Microbiol. 38, 423-427.
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20. Panthee, S., Paudel, A., Hamamoto, H., and Sekimizu, K., 2017. Advantages of the Silkworm
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as an Animal Model for Developing Novel Antimicrobial Agents.Front. Microbiol. 8, 373.
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21. Prajapati, R.A., Raol, B.V., 2014. The study on the efficacy of some herbal extracts for the
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control of dental caries pathogen-Streptococcus mutans. Int. J. Pharm. Sci. Health Care. 1,
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22. Ramalingam, K., Amaechi, B.T., 2018. Antimicrobial effect of herbal extract of Acacia
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Arabica with Triphala on the biofilm forming cariogenic microorganisms. J. of Ayur. and Int.
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Med. https://doi.org/10.1016/j.jaim.2018.01.005.
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23. Ramalingam, K., Amaechi, B.T., Rawls, H.R., Lee, V.A., 2012. Antimicrobial activity of
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nanoemulsion on cariogenic planktonic and biofilm organisms. Arch Oral Biol. 57, 15-22.
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24. Saxena, S., Lakshminarayan, N., Gudli, S., Kumar, M., 2017. Anti-Bacterial Efficacy of
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Terminalia chebula, Terminalia bellirica, Embilica officinalis and Triphala on Salivary
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Streptococcus mutans Count – A Linear Randomized Cross Over Trial, Journal of Clinical
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and Diagnostic Research.11(2), ZC47 - ZC51.
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25. Shetty, P.J., Hegde, V., Gomes, L., 2014. Anticandidal efficacy of denture cleansing
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tablet, Triphala, Aloe vera, and Cashew leaf on complete dentures of institutionalized
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elderly. J. Ayurveda Integr Med. 5, 11-4.
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26. Sonis, S.T., Clark, W.B., Shklar, G., 1978. Chlorhexidine-induced lingual keratosis and
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dysplasia in rats. J.Periodontol. 49(11), 585-91. 27. Teixeira, P.C., Leite, G.M., Domingues, R.J., Paul, J.S., Gibbs, A., Ferreira, J.P., 2007.
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Antimicrobial effects of a microemulsion and a nanoemulsion on enteric and other pathogens
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and biofilms. International Journal of Food Microbiology.118, 15–19.
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28. The Ayurvedic Pharmacopoeia of India, 2016. First edition, published by Pharmacopoeia Commission of Indian Medicine and Homeopathy.
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29. Thomas, B., Shetty, S., Vasudeva, A., Shetty, V., 2011. Comparative evaluation of
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Antimicrobial Activity of Triphala and commercially available toothpastes: An in-vitro
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study. Int. J. Public Health Dent. 2, 8-12.
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30. Usui, K., Nishida, S., Sugita, T., Ueki, T., Matsumoto, Y., Okumura, H., and Sekimizu, K.
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2016.Acute oral toxicity test of chemical compounds in silkworms. Drug Discov. Ther. 10,
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57–61. 31. Welihinda, J., Ariyawansa, H.A., Wickramasinghe, R., 2006. Comparison of the diuretic
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effects of frusemide and the KaraviPanchaka Ayurveda decoction. Ceylon Med J. 51(3):93-7.
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32. Zhang, X. L., Xue, R.Y., Cao, G. L., Pan, Z. H., Zheng, X. J., and Gong, C. L.
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2015. Silkworms can be used as an animal model to screen and evaluate gouty therapeutic
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drugs. J. Insect Sci. 12, 4.
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Fig. 1. FTIR spectrum of i. Emblica officinalis decoction, ii. Terminalia chebula decoction, iii.
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Terminalia bellericadecoction, iv. Acacia arabica decoction, v. Mixed decoction
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Fig. 2. Photograph of the zone of inhibition of the various microorganisms against the different
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decoctions. A-F: Zone of inhibition of Streptococcus mutans against Embilica officinalis
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Decoction (A), Terminalia chebula Decoction (B), Terminalia bellerica Decoction (C), Acacia
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arabica Decoction (D), Mixed Decoction (E), Chlorohexidine (F). G-L: Zone of inhibition of
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Lactobacillus casei against Embilica officinalis Decoction (G), Terminalia chebula Decoction
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(H), Terminalia bellerica Decoction (I), Acacia arabica Decoction (J), Mixed Decoction (K),
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Chlorohexidine (L). M-R: Zone of inhibition of Actinomyces viscosus against Embilica
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officinalis Decoction (M), Terminalia chebula Decoction (N), Terminalia bellerica Decoction
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(O), Acacia arabica Decoction (P), Mixed Decoction (Q), Chlorohexidine (R).
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Fig. 3. Graph representing the zone of inhibition of the various microorganisms against the
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different decoctions.
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Fig. 4. Streptococcus mutans count after treatment with decoctions at i. 1, ii. 5, iii. 15, iv. 30 and
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v. 60 minutes. (Eo-D: Embilica officinalis Decoction,Tc-D: Terminalia chebulaDecoction,Tb-D:
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Terminalia bellericaDecoction, Aa-D: Acacia arabica Decoction, MD: Mixed Decoction, PC:
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Positive Control, NC: Negative Control).
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Fig. 5.Lactobacillus casei count after treatment with decoctions at i. 1, ii. 5, iii. 15, iv. 30 and v.
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60 minutes.
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Fig. 6.Actinomyces viscosus count after treatment with various decoctions at i. 1, ii. 5, iii. 15, iv.
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30 and v. 60 minutes.
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Fig. 7.Combined microbial count after treatment with decoctions at i. 1, ii. 5, iii. 15, iv. 30 and v.
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60 minutes.
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Fig. 8. Microbial count after the adherence assay using drop plate method in a. Streptococcus
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mutans, b. Lactobacillus casei, c. Actinomyces viscosus and d. combined bacteria. (Eo-D-
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Embilica officinalis Decoction,Tc-D-Terminalia chebulaDecoction,Tb-D-Terminalia
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belliricaDecoction, Aa-D- Acacia arabica Decoction, MD- Mixed Decoction, PC- Positive
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Control, NC- Negative Control).
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Fig. 9. Photograph of Bombyx mori after 24 hours of treatment. A, B, C- Control toxicity study
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with mixed decoction, chlorohexidine and distilled water; D, E, F- S. mutans; G, H, I- L. casei; J,
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K, L- A. viscosus; M, N, O- combined microbe infection assay using mixed decoction,
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chlorohexidine and distilled water respectively.
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Fig.10. Control graph represents the surviving population (%) of B. mori worms after 24 hours of
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administration of the 50% diluted decoctions and chlorohexidine (control toxicity study),b.
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Streptococcus mutans, c. Lactobacillus casei, d. Actinomyces viscosusand e. combination of
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allthree micro-organisms infection assay with Bombyx mori. (Eo-D: Embilica officinalis
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Decoction,Tc-D: Terminalia chebulaDecoction,Tb-D: Terminalia bellericaDecoction, Aa-D:
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Acacia arabica Decoction, MD: Mixed Decoction, PC: Positive Control, NC: Negative Control)
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S1878-8181(18)30674-1
DOI:
https://doi.org/10.1016/j.bcab.2019.101026
Article Number: 101026 Reference:
BCAB 101026
To appear in:
Biocatalysis and Agricultural Biotechnology
Received Date: 6 October 2018 Revised Date:
21 November 2018
Accepted Date: 19 December 2018
Please cite this article as: Sneha, U., Karthikeyan, R., Bactericidal activity of ayurvedic formulation against cariogenic microorganisms, Biocatalysis and Agricultural Biotechnology (2019), doi: https:// doi.org/10.1016/j.bcab.2019.101026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Bactericidal activity of ayurvedic formulation against cariogenic microorganisms
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Authors Names: Sneha U and Karthikeyan Ramalingam
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Affiliations: School of Life Sciences, B.S. Abdur Rahman Crescent Institute of Science and
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Technology, Chennai, India
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1. Sneha U
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Research scholar
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School of Life Sciences, B.S. Abdur Rahman Crescent Institute of Science and Technology
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GST Road, Vandalur, Chennai -600 048, India
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Phone: +91 44 22759223; +91 44 22751347; +919940323240
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Corresponding Author Information:
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2. Dr. Karthikeyan Ramalingam
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Associate Professor
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School of Life Sciences, B.S. Abdur Rahman Crescent Institute of Science and Technology
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GST Road, Vandalur, Chennai -600 048, India
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Phone: +91 44 22759223; +91 44 22751347; Mobile +917502534668
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Email: [email protected]
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Bactericidal activity of ayurvedic formulation against cariogenic microorganisms Abstract Dental caries is an infectious disease characterized by formation of biofilm on the teeth.
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It is a major health concern worldwide and can be prevented.The dried powders of Acacia
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arabica, Terminalia chebula, Terminalia bellerica and Emblica officinalishave the potential to
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cure oral diseases and these four plant powders have been used in traditional tooth powder
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formulas in India for more than 100 years. The objective of the present study was to investigate
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the antimicrobial effect of an aqueous extract of these four plant materials against the cariogenic
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microorganisms. The Minimum Inhibitory Concentration, Minimum Bactericidal Concentration,
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kinetics of killing, and adherence assay of the aqueous plant extracts against the cariogenic
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microorganisms were determined. The results showed that the combined decoction of plant
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extracts had a high bactericidal activity against all the biofilm forming cariogenic
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microorganisms tested. Toxicity studies and infection assay on Bombyx mori proved that the
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plant decoctions were not toxic. These assays suggest that the combined decoction could act as a
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potential mouthwash effective against dental caries.
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Keywords: Cariogenic microorganisms; Time-kinetics; adherence assay; Triphala; Acacia
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Arabica; toxicity studies.
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1. Introduction
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Dental caries is a worldwide problem affecting millions of people. It is a microbial infectious
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disease caused due to the demineralization and remineralization of the tooth surface. The
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microbes convert sucrose to acids which destroy the tooth enamel by decalcification(Badria and
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Zidan, 2004). The chief bacteria which cause tooth decay are Streptococcus mutans,
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Lactobacillus casei and Actinomyces viscosus(Jenkinson and Lamont, 2005).
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Mouthwash is a potential inhibitor of dental caries and it containstriclosan, cetylpyridinium
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chloride, zinc citrate or chlorohexidine. Though they are effective, they have certain side effects
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like staining of teethand development of antimicrobial strains(Ramalingam and Amaechi, 2018).
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A number of plants like orange tree, lime tree, neem plants have been used for years as
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chewing sticks.Literature has shown that neem has antimicrobial and therapeutic effects
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suggesting its potential to be used as an endodontic irrigant(Biswas et al., 2002). Murray et 2
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al.(2008)also suggested that Morindacitrifolia juice can be formulated for use as an intracanal
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irrigant.Traditional plants like Acacia arabica (bark), Terminalia chebula(fruits), Terminalia
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bellerica(fruits) and Emblica officinalis (fruits) have the potential to treat dental caries and have
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been used traditionally in India for more than 100 years(Chouhan, 2008). A combination of T.
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chebula, T.bellericaand E. officinalis, has a number of potential uses which include free radical
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scavenging, antioxidant, anti-inflammatory, dental caries prevention, immunomodulating,
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appetite stimulation, gastric hyperacidity reduction, chemoprotective,antipyretic, antistress,
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adaptogenic,
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hepatoprotective,
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chemopreventive effects (Baliga et al., 2012; Shetty et al., 2014).The advantages of using herbal
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alternatives are cost-effectiveness,easy availability, low toxicity,increased shelf lifeand lack of
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microbial resistance (Abascal and Yarnell, 2002; Balakrishnan et al., 2018).
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anticariogenic,
anticandidal,
hypoglycemic,
antimutagenic, anticancer,
wound
healing,
radioprotective,
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antibacterial,
and
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analgesic,
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The objective of the present study was to investigate the antimicrobial effect of a mouth wash prepared from Triphala in combination with Acacia arabica.
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2. Materials and Methods
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2.1.Materials
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The dried powders Acacia arabica (bark), Terminalia chebula(fruits), Terminalia
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bellerica(fruits) and Emblica officinalis (fruits)has been used in traditional tooth powder
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formulation in India for more than 100 years and were purchased from the local traditional stores
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in Chennai. The powders were of pharmacopoeia standards with reference to “The Ayurvedic
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Pharmacopoeia of India”. These powders were weighed and prepared into individual decoctions
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as well as in mixed ratio of 7:1:1:1 (A. arabica (70%), T. chebula(10%), T. bellerica(10%), E.
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officinalis (10%)) (Karthikeyan and Amaechi, 2018).The three biofilm forming cariogenic
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bacteria used in this study Streptococcusmutans(ATCC 25175),Lactobacillus casei(ATCC 393)
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and Actinomyces viscosus(ATCC 15987) were purchased from the American Type Culture
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Collection (ATCC).The media used for the growth of these bacteria were Brain Heart Infusion
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(BHI) agar and BHI broth purchased from Himedia.
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2.2.Preparation of Decoction
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Herbal decoction was prepared as per the protocol given in Sarangdhar samhita (Tripathi.,
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2001). All the individual powders as well as the combined powders were mixed with distilled 3
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water and boiled for 5 mins on an induction stove till a strong decoction was formed. The overall
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volume reduced to 1/8th of its original volume.After the decoction was prepared, it was cooled to
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room temperature. The decoctions were then filtered and centrifuged at 4000 rpm for 10 minutes
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to get a clear solution. The supernatant was stored in falcon tubes for further analysis.
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2.3.Fourier Transform Infrared Spectroscopy (FTIR) analysis
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FTIR analysis is an important technique used to detect the different bonds present in a
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compound. The decoctions were dried and the powder was subjected to FTIR analysis in a Jasco
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FTIR 6300 spectrometerwith a scan range from 400 to 4000 cm-1having a resolution of 4cm-1to
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detect different functional groups present in the sample. The different peaks of the various
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decoction samples were observed and noted.
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2.4.Antibacterial activity
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The antibacterial activity of all the individual and combined decoctions was assayed against
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S. mutans, L. casei and A. viscosus (107 CFU/ml) using the agar well diffusion method. The
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Brain Heart Infusion (BHI) agar plates were spread with 100 µl of the culture and a well was cut
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using the cork borer. One hundred microliters of the decoctions were added intothe well and the
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plates were incubated overnight at 37°C.The assay was conducted in triplicate.
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2.5.MIC and MBC
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Plant extract was serially diluted with S. mutans cell suspension (107 CFU/ml) and was
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incubated overnight at 37ºC. The lowest concentration showing no growth was recorded as the
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minimum inhibitory concentration (MIC). For Minimum Bactericidal Concentration (MBC)
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testing, aliquots were plated onto Brain Heart Infusion (BHI) agar and incubated overnight at
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37°C. The lowest concentration showing 99.9% killing was recorded as the MBC.
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Chlorohexidine was used as positive control.MIC and MBC assays were conducted for L. casei
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and A. viscosusas well.The assays were conducted in triplicate(Karthikeyan et al., 2011;
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Ramalingam et al., 2012).
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2.6.Time kinetics
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Overnight cultures of S. mutans, L. casei, A. viscosusand mixedcultures of the three strains
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were added to plant decoctions inmicrotiter plates. Same concentrations of chlorohexidine were
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used as positive control. Following addition of the bacterial culture, 100 µl sample was retrieved
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from each plate at 1, 5, 15, 30 and 60 min and serially diluted (10-1– 10-4). The bacterial count
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was estimated using plate count method for all the dilutions(Karthikeyan et al., 2011; 4
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Karthikeyan and Amaechi, 2018).The dilution which showed countable number of microbes was
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noted along with the count of the bacteria.The experiment was conducted in triplicate.
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2.7.Adherence assay Overnight bacterial cultures containing 107 CFU/ml of S. mutans, L. casei, A. viscosusand
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mixed cultures of the three strains were added to BHI broth containing plant decoctions
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individually and in combination in the ratio 1:1. The tubes were incubated aerobically(to
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simulate conditions in the mouth) at 37°C for 24h at an angle of 30°(Ramalingam et al., 2012;
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Karthikeyan and Amaechi, 2018). Chlorohexidine 0.2% (v/v) was used as positive control. The
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contents of the tubes were poured out and the tubes were washed with distilled water. One
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milliliter of autoclaved distilled water was then added to all the tubes and the tubes were
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vortexed for a minute. Subsequently dilutions were made from this starting from 10-1 upto 10-4
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for all the samples. The bacterial count was estimated at each dilution for all the samples by plate
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count technique. The dilution which showed countable number of microbes was noted along with
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the count of the bacteria.All experiments were carried out in triplicate.
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2.8.Bombyx mori Toxicity Infection Assay
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As an invertebrate model, silkworm model is characterized by its convenience, low cost, no
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ethical issues. The presence of conserved immune response and similar pharmacokinetics
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compared to mammals make silkworm infection model suitable to examine the therapeutic
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effectiveness of antimicrobial agents (Panthee et al., 2017).Bombyx mori collected from
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sericulture units in Alangayam, Tamil Nadu was acclimatized in the lab and fed with mulberry
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leaves.
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For control studies,Bombyx mori worms (5 per set) were injected with 200 µL of the plant
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extract diluted 50% with ultrapure water. Two hundred microliter of chlorohexidine was used as
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positive control and as a negative control 200 µL of sterile distilled water was injected into the
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worms. They were incubated for a day at room temperature. The number of worms surviving
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were noted after 24 hours. The experiment was conducted in triplicate (Zhang et al., 2015; Usui
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et al., 2016).
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For carrying out the infection assay, the worms were first injected with 1 unit of bacterial
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culture S. mutans. After 15 mins, they were injected with 2 units of the plant decoctions
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individually or in combination. For the positive control, 2 units of chlorohexidine was injected
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and for negative control, 2 units of sterile distilled water was injected into the worms. They were 5
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incubated for a day at room temperature. The number of worms surviving were noted after 24
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hours.This assay was repeated with L. casei, A. viscosus and combination of all 3 bacteria. The
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experiment was conducted in triplicate (Zhang et al., 2015; Usui et al., 2016).
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All the data was statisticallyanalyzed using one-way ANOVA. Criterion for statistical
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significance was defined as P < 0.05.
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3. Results and Discussion
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3.1. Preparation of Decoction
Acacia arabica, Terminalia chebula, Terminalia bellericaand Emblica officinalis decoctions
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were prepared individually (Welihinda et al. 2006). A combined decoction ofAcacia arabica,
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Terminalia chebula, Terminalia bellericaand Emblica officinalis was preparedin the ratio of
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7:1:1:1 respectively(Karthikeyan and Amaechi, 2018).
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3.2.Fourier Transform Infrared Spectroscopy (FTIR) analysis
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Figure 1 represents the various FTIR spectra of the different decoctions.FTIR spectrum of
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decoction of Emblica officinalis showed a major peak at 3218.61 cm-1 which indicates the
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presence of phenols and alcohols. The other peaks at 1696.12 cm-1, 1691.96 cm-1, indicates the
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presence of carboxylic acids, peak at 1402.96 cm-1 indicates alkanes, peaks at 1216.86 cm-1,
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1032.69 cm-1 represent esters or ethers, the other peaks at 867.81 cm-1, 800.314 cm-1, 621.931
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cm-1, 617.793 cm-1 represent alkyl halides.FTIR spectrum of decoction of Terminalia chebula
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showed a major peak at 3200.29 cm-1 which indicates the presence of phenols and alcohols. The
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other peaks at 1696.09 cm-1, 1598.7 cm-1, indicates the presence of carboxylic acids, peak at
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1328.71 cm-1 indicates alkanes, peaks at 1193.72 cm-1, 1031.73 cm-1 and 632.637 cm-1 represent
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alkyl halides.FTIR spectrum of decoction of Terminalia belliricashowed a major peak at 3231.16
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cm-1 which indicates the presence of phenols and alcohols. The other peaks at 1696.09 cm-1,
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1602.66 cm-1 indicate the presence of carboxylic acids, peak at 1327.76 cm-1 indicates alkanes,
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peaks at 1186.04 cm-1, 1029.8 cm-1 and 417.513 cm-1 represent alkyl halides, and peak at 698.105
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cm-1 represents alkynes.FTIR spectrum of decoction of Acacia arabica showed a major peak at
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3219.68 cm-1 which indicates the presence of phenols and alcohols. The other peaks at 1606.45
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cm-1, 1442.49 cm-1 indicate the presence of carboxylic acids, peaks at 1321 cm-1, 1209.16 cm-1,
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1030.77 cm-1 and 768.494 cm-1 represent alkyl halides.FTIR spectrum of decoction of mixed
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decoction of all 4 plants showed a major peak at 3210.9 cm-1 which indicates the presence of
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phenols and alcohols. The peak at 1697.05 cm-1 indicates the presence of aldehydes, peak at
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1599.66 cm-1 represents amides, peaks at 1334.6 cm-1, 1199.61 cm-1, 1031.73 cm-1 and 616.145
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cm-1 represent alkyl halides. FTIR analysis of the Triphala showed the presence of different functional groups ranging
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from O-H alcohol (3200-3600 cm-1), C-H alkane (2850-3000 cm-1), =C-H aldehyde (2820-2850
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cm-1), acyclic ketone (1705-1725 cm-1), N-H amide (1550-1640 bending), C=C aromatic (1400-
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1600 cm-1), C-N amine (1080-1360 cm-1), C-F alkyl Halide (1000-1400 cm-1), =C-H alkene
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(675-1000 cm-1) as functional groups(Amala and Jeyaraj, 2014).
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3.3.Antibacterial activity
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The antimicrobial activity of plant decoctionswas checked against Streptococcus mutans,
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Lactobacillus caseiand Actinomyces viscosus. It wasquantitatively assessed by measuring the
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inhibition zone. Plant extract was effective with the inhibition zones ranging from 13 to 24 mm.
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The mixed decoction had a maximal zone of inhibition of 20 mm, 24 mm and 19 mm when
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compared to chlorohexidine which showed a zone of inhibition of 18 mm, 21 mm and 19 mm
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against Streptococcus mutans, Lactobacillus caseiand Actinomyces viscosusrespectively (p <
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0.05). Hence, combined decoction is a better antibacterial agent in comparison to chlorohexidine.
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Figure 2and 3 represent the photograph and graph of the zone of inhibition of the different
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microbes against the different decoctions.
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Invitro antimicrobial activity of Triphala (aqueous extract)with Staphylococcus aureus,
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Pseudomonas aeruginosa and Klebsiella pneumoniae has been reported to have inhibitory zone
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at 14mm, 10.75mm, 10mm(Amala and Jeyaraj, 2014).Gupta et al.(2014) has reported that the
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combined aqueous extract of Terminalia chebula, Emblica officinalis and Terminalia bellerica
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showed higher zone of inhibition compared to 0.2% chlorohexidine.Thomas et al. (2011) found
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the mean inhibitionzone for the aqueous extract of Triphala(combination of Emblica officinalis,
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Terminalia chebula and Terminalia bellerica)at 50%, 25%and 12.5% against S. mutans(MTCC
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strain) to be 30 mm, 28 mm, and24 mm respectively. The mean inhibition zone for the
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aqueousextract of Triphala at 50%, 25% and 12.5% against clinicalisolates of S. mutanswas
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found to be 34 mm, 30 mm, and28 mm, respectively.Prajapathi and Raol (2014) found the
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aqueous extract ofTriphala to inhibit S. mutans. The mean inhibition zone was17 mm against
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MTCC strains and 19 mm against clinicalisolates.
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3.4.MIC and MBC
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The MIC assay was performed for all the three bacteria. All the three bacteria showed
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inhibition upto the 3rd well (p < 0.05). So upto 3 fold dilution there was inhibition. Hence the
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MIC was at 1.25 mg/ml.
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MBC was performed from 1-6 wells for all the samples along with negative control and media
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control. The concentration up to which there was 99.9% reduction in bacterial load was
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considered to be as MBC. The concentration of MBC was 2.5 mg/ml(p < 0.05).
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Triphala showed minimal inhibitory concentration and minimal bactericidal concentration at
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6.25% for MTCC strain of S.mutans and 3.12% for clinical isolate of S.mutans(Thomas et al.,
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2010).MICs and MBCs of the colloidal solutions containing ZnO, CuO, TiO2 and Ag
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nanoparticles were comparable to each other and all were significantly lower than that of the
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chlorhexidine mouthrinse against both S. mutans and S. sangius(Ahrari et al., 2015).
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3.5.Kinetics Study
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Kinetics study at time points 1, 5, 15, 30 and 60 minutes was performed for all the bacteria-
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individually and in combination. Throughout the time period, the number of colonies in the
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decoction treated samples was much lesser when compared to the negative control(p < 0.05). The
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number of bacterial colonies increased rapidly from 1 minute to 60 minutes in the negative
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control. In all the treated samples, the number of microbes was gradually increased from 1
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minute to 15 minutes followed by gradual reduction(p < 0.05). The positive control did not
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contain any bacterial colonies in all the cases.Figures 4-7represent the graphs of the
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Streptococcus mutans, Lactobacillus casei, Actinomyces viscosus and combination of all three
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bacterial cultures after treatment with various decoctions.The combined decoction
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Nanoemulsions have been found to have bactericidal properties against Grampositiveand
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enteric pathogen species in various earlier reports(Teixeira et al., 2007; Hamouda et al., 1999,
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2001).Greater killing (49–94.73%) of S. mutans was observed with mixed herbal powder extract
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containing methanolic extract of triphala and Acacia arabica at 60 min when compared with
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chlorohexidine (36.56–65.93%) during kinetics study (Karthikeyan and Amaechi, 2018).
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3.6.Adherence Assay
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Adherence assay was performed with all the 3 bacteria bothindividually and as a consortium-
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Streptococcus mutans, Lactobacillus casei and Actinomyces viscosus. The number of bacterial
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colonies of all dilutions was noted the next day. In all the 4 cases, the number of bacterial
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colonies when treated with the various decoctions showed much lesser bacterial count when 8
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compared to the negative control which was untreated(p < 0.05). The number of bacterial
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colonies after being treated with the mixed decoction was 1000-fold lesser than the negative
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control bacterial count in all the cases. The positive control showed absence of bacteria in all the
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cases(p < 0.05). Figure 8 represents the graphs of bacterial count of various treated samples. Glass adherence assay used in the present study is an effective model for assessing sucrose
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dependent bacterial adhesion (Nostro et al., 2004).More inhibition of S. mutans adherenceby
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mixed herbal powder extractcontaining methanolic extract of triphala and Acacia Arabica (up to
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85.64%) at 100 µg/ml concentration has been reported when compared tochlorohexidinewhich
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showed inhibition up to 73.21% (Karthikeyan and Amaechi, 2018).
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3.7.Bombyx mori infection assay
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This study is the first study in which plant decoctions have been tested against cariogenic
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microbes in vivo using silk worm as a model organism. In control studies, the photographs of the
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worms after 24 hours of treatment are shown in figure 9. Figure 10 represents the graph
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indicating the percentage of surviving population of B. mori worms after 24 hours of
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administration of the 50% diluted decoctions and chlorohexidine.
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In the control, the worms given only plant decoctions survived 100%(p < 0.05). The worms
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injected with distilled water also survived 100%. The worms injected with chlorohexidine
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survived only 60%(p < 0.05). This proved that the plant decoctions were not toxic whereas the
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chlorohexidine was toxic.Side effects of chlorohexidine like altered taste perception, metallic
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taste, and staining of teeth have been reported earlier (Flotra et al., 1971).Chlorohexidine shows
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cytotoxicity and even causes dysplasia of the oral mucosa (Sonis et al., 1978; Cabral et al., 2007;
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De Souza et al., 2017).
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In S. mutans infection assay, worms treated with Terminalia bellerica and mixed
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decoction showed higher survival rates (65%) compared to untreated control as well as worms
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treated with chlorohexidine- both of which showed only 45% survival(p < 0.05). This showed
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that treatment with mixed decoction was better than chlorohexidine.In all the other bacterial (L.
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casei, A. viscosus and mixed culture of all 3 bacteria) infections, the plant decoctions showed
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better survival rate than the positive control(p < 0.05).Saxena et al., (2017) had reported that
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the mean colony forming units of S. mutans with Triphala was significantly reduced at 5 minutes
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when tested on human trials. The ability of these rinses to reduce plaque, freshen breath, to
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prevent or control tooth decay, to reduce gingivitis, to reduce the speed that tartar forms on the
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teeth, or to produce a combination of these effects (Kornman, 1986).
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4. Conclusion Thus the aqueous plant extract prepared from the plants- Acacia arabica, Terminalia
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chebula, Terminalia bellerica and Emblica officinalis was found to be effective against the
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common dental caries causing bacteria- Streptococcus mutans, Lactobacillus casei and
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Actinomyces viscosus.In this study, antibacterial activity, MIC, MBC, kinetics study, adherence
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assay and toxicity studies was performed. Commercially available chlorohexidine seemed to be
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an effective antibacterial agent but wascytotoxicand led to reduced survival of B. mori. The
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combined plant decoction gave 99.9% reduction in the total amount of bacteria while proving to
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be completely non-toxic to B. mori.
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5. Funding
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This work was supported by The Ministry of Ayurveda, Yoga and Naturopathy, Unani, Siddha
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and Homoeopathy (AYUSH)-EMR project(Sanction order No. Z.28015/209/2015-HPC) and
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DST Science and Engineering Research Board (SERB) [Grant number- SERB/LS-267/2014].
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1. Abascal, K., Yarnell, E., 2002. Herbs and drug resistance Part 2 - Clinical implications of
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2. Ahrari, F., Eslami, N., Rajabi, O., Ghazvini, K., and Barati, S., 2015. The antimicrobial 300
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different nanoparticles applied as mouthwashes. Dent. Res. J.12(1), 44–49. 302
3. Amala, V.E., Jeyaraj, M., 2014. Determination of Antibacterial, Antifungal, Bioactive
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Constituents of Triphalaby FT-IR and GC-MS Analysis, Int J Pharmacy and Pharmaceutical
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4. Badria, F.A., Zidan, O.A., 2004. Natural products for dental caries prevention. J. Med. Food. 7, 381-4. 5. Balakrishnan, B., Nair, R., Perayil, J., Fenol, A., Menon, L., Vyloppillil, R., Bhaskar, A.,
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6. Baliga, M.S., Meera, S., Mathai, B., Rai, M.P., Pawar, V., Palatty, P.L.,2012. Scientific
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13. Hamouda, T., Hayes, M.M., Cao, Z., 1999. A novel surfactant nanoemulsion with broadspectrum sporicidal activity against Bacillus spores. J. Infect. Dis.180, 1939–1949. 14. Hamouda, T., Myc, A., Donovan, B., Shih, A.Y., Reuter, J.D., Baker, J.R.,2001. A novel
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24. Saxena, S., Lakshminarayan, N., Gudli, S., Kumar, M., 2017. Anti-Bacterial Efficacy of
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Terminalia chebula, Terminalia bellirica, Embilica officinalis and Triphala on Salivary
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30. Usui, K., Nishida, S., Sugita, T., Ueki, T., Matsumoto, Y., Okumura, H., and Sekimizu, K.
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32. Zhang, X. L., Xue, R.Y., Cao, G. L., Pan, Z. H., Zheng, X. J., and Gong, C. L.
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drugs. J. Insect Sci. 12, 4.
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Fig. 1. FTIR spectrum of i. Emblica officinalis decoction, ii. Terminalia chebula decoction, iii.
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Terminalia bellericadecoction, iv. Acacia arabica decoction, v. Mixed decoction
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Fig. 2. Photograph of the zone of inhibition of the various microorganisms against the different
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decoctions. A-F: Zone of inhibition of Streptococcus mutans against Embilica officinalis
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Decoction (A), Terminalia chebula Decoction (B), Terminalia bellerica Decoction (C), Acacia
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arabica Decoction (D), Mixed Decoction (E), Chlorohexidine (F). G-L: Zone of inhibition of
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Lactobacillus casei against Embilica officinalis Decoction (G), Terminalia chebula Decoction
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(H), Terminalia bellerica Decoction (I), Acacia arabica Decoction (J), Mixed Decoction (K),
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Chlorohexidine (L). M-R: Zone of inhibition of Actinomyces viscosus against Embilica
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officinalis Decoction (M), Terminalia chebula Decoction (N), Terminalia bellerica Decoction
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(O), Acacia arabica Decoction (P), Mixed Decoction (Q), Chlorohexidine (R).
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Fig. 3. Graph representing the zone of inhibition of the various microorganisms against the
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different decoctions.
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Fig. 4. Streptococcus mutans count after treatment with decoctions at i. 1, ii. 5, iii. 15, iv. 30 and
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v. 60 minutes. (Eo-D: Embilica officinalis Decoction,Tc-D: Terminalia chebulaDecoction,Tb-D:
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Terminalia bellericaDecoction, Aa-D: Acacia arabica Decoction, MD: Mixed Decoction, PC:
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Positive Control, NC: Negative Control).
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Fig. 5.Lactobacillus casei count after treatment with decoctions at i. 1, ii. 5, iii. 15, iv. 30 and v.
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60 minutes.
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Fig. 6.Actinomyces viscosus count after treatment with various decoctions at i. 1, ii. 5, iii. 15, iv.
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30 and v. 60 minutes.
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Fig. 7.Combined microbial count after treatment with decoctions at i. 1, ii. 5, iii. 15, iv. 30 and v.
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60 minutes.
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Fig. 8. Microbial count after the adherence assay using drop plate method in a. Streptococcus
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mutans, b. Lactobacillus casei, c. Actinomyces viscosus and d. combined bacteria. (Eo-D-
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Embilica officinalis Decoction,Tc-D-Terminalia chebulaDecoction,Tb-D-Terminalia
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belliricaDecoction, Aa-D- Acacia arabica Decoction, MD- Mixed Decoction, PC- Positive
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Control, NC- Negative Control).
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Fig. 9. Photograph of Bombyx mori after 24 hours of treatment. A, B, C- Control toxicity study
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with mixed decoction, chlorohexidine and distilled water; D, E, F- S. mutans; G, H, I- L. casei; J,
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K, L- A. viscosus; M, N, O- combined microbe infection assay using mixed decoction,
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chlorohexidine and distilled water respectively.
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Fig.10. Control graph represents the surviving population (%) of B. mori worms after 24 hours of
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administration of the 50% diluted decoctions and chlorohexidine (control toxicity study),b.
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Streptococcus mutans, c. Lactobacillus casei, d. Actinomyces viscosusand e. combination of
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allthree micro-organisms infection assay with Bombyx mori. (Eo-D: Embilica officinalis
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Decoction,Tc-D: Terminalia chebulaDecoction,Tb-D: Terminalia bellericaDecoction, Aa-D:
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Acacia arabica Decoction, MD: Mixed Decoction, PC: Positive Control, NC: Negative Control)
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