Preliminary report

Assessing the Efficacy of Herbal Mouthwash Versus Commercial Products

in Controlling Oral Microbiota

Rohankumar Mistry (301280696)

Luis E Sarabia (301267675)

BI 206-703

Professor: Natasha Sigh

Centennial College

February 25th, 2024

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 Table of contents

Title Page ...........……………………………………………………………………………. 1

Table of Contents……………………………………………………………………………. 2

1. Introduction………………………………………………………………………………... 3

1.1 Background…………….………………………………………..………………… 3

1.2 Rationale…………….………………………………………..…………….……… 4

1.3 Objectives…………….………………………………………..…...……………… 4

1.4 Hypothesis…………….………………………………………..……………..…… 5

1.5 Methodology applied…………….………………………..………………………. 5

1.6 Bacterial culture used …………….………………………..……………..……… 6

1.7 Media to be used …………….………………………..…………………….…… 6

2. Methodology………………………………………………………………………………... 7

2.1 Project Material list……………………………………..…………………….…… 7

2.2 Experimental Design…………….………………………..…………….………… 9

2.3 Controls…………….………………………………………..……………….…… 11

3. Reference…………….………………………………………..…………………..……… 12

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 INTRODUCTION

1.1 Background

Views of consumers have changed significantly in support of organic and herbal

remedies in recent years. These days, oral care products are also subject to this
movement. Kitchen pantry items like cinnamon, clove, and mint have all shown interest
alternatives to common ingredients in dental care products. These organic substances
offer potential advantages for tooth health in addition to improving flavor. Their culinary
applications typically makeing them valuable. Mint is well known for its stimulating flavor
and aroma and has long been known to have germicidal abilities (Elementa Nano Silver
Xylitol Dental Mints, n.d.). It may organically destroy oral bacteria, which is why
toothpaste, mouthwashes, and dental mints usually contain it. Eugenol, ingredient with
anesthetic, antibacterial, and antifungal qualities, gives cloves their comforting, fragrant
perfume (Medical News Today, 2018). Clove oilis ancient toothache cure because
eugenol help with gum infections and dental pain. Known for its spicy and sweet smell,
cinnamon contains number of health-promoting ingredients, including eugenol,
cinnamaldehyde, and cinnamic acid (Yanakiev, 2020). These components provide
cinnamon with its antimicrobial, anti-inflammatory, and antibacterial qualities, perhaps
becoming it invaluable instrument for maintaining oral health. study of natural
ingredients included in dental care products, such as mint, cinnamon, and cloves, has
scientific importance. Yanakiev's (2020) review sheds light on various applications and
effects of cinnamon in oral health, highlighting spice's potential in dentistry. Meanwhile,
usage of clove oil for toothaches highlights its long history of use as natural pain reliever
(Medical News Today, 2018). commercial dental care products like peppermint-infused
Elementa Nano Silver Xylitol Dental Mints harness germkilling qualities of mint to help
prevent cavities and gum infections. In considering this, our study's objective is to
evaluate effectiveness of herbal mouthwash extract made from cinnamon, cloves, and
mint against well-known dental hygiene products. To produce concentrated extracts
from these organic components, we will employ Soxhlet extraction, technique that is well
known for its effectiveness in extracting chemicals from botanical materials. Our
methodical approach is to assess these extracts' capacity to modify oral microbiota,
concentrating on two common oral bacteria: Lactobacillus acidophilus and
Streptococcus mutans. benefits of using well-known antibacterial properties of cloves,
cinnamon, and mint in herbal mouthwash extracts are not yet understood. Our goal is to
fill knowledge gap and offer helpful details on possible use of natural extracts in oral
hygiene through careful experimentation and data analysis. Our research may provide
knowledge that oral health professionaland consumers need to make wise choices
regarding oral hygiene habits. Ultimately, we that our evidence-based research and

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support of natural dental care alternatives will enhance oral health outcomes for people
worldwide.

1.2 Rationale

Health and Hygiene factor: Oral Care is essential for overall well-being; balance
of oral microorganisms can be unpredictable and varies among individuals. As this
balance fluctuates, this can lead to range of problems such as cavities, halitosis, and
gum diseases. Assessing efficacy of mouthwash choices holds direct relevance for
public health.
Natural alternative: Nowadays, Consumers are increasingly drawn to natural or
herbal substitutes in their quest for oral care products, often perceiving them as safer or
more effective than industrial and artificial ingredients. This research aims to empower
consumers with scientific evidence to guide their decision-making process when
selecting between herbal and commercial mouthwash options. By evaluating and
comparing antimicrobial efficacy of these choices, individuals can make more informed
decisions aligned with their oral health preferences or for second evaluation in further
future.

1.3 Objectives

The goal of this extensive study is to give people full understanding of

antibacterial efficacy of dental products and natural extracts, empowering them to make
decisions that will help them maintain. their best possible oral health. studys
investigation of viable alternatives for oral care is given new perspective using natural
extracts.
The study evaluates comparative effectiveness of herbal mouthwash, comprising
cloves, peppermint, and cinnamon, alongside comercial mouthwash product, in
controlling growth of Streptococcus mutans and Lactobacillus acidophilus. Additionally,
it examines known antibacterial efficacy of individual components (cloves, peppermint,
cinnamon) as theoretical framework, while also investigating practical research of their
combination within herbal mouthwash against Streptococcus mutans and Lactobacillus
acidophilus. Moreover, study explores potential undisclosed benefits of this mouthwash
properties.

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 1.4 Hypothesis

herbal mouthwash extract will demonstrate similar or superior antibacterial

activity compared to commercial mouthwash product oppose to S. mutans and L.
acidophilus; Increasing concentrations of herbal mouthwash will result in decrease in
bacterial growth; and Null hypothesis there is no significant difference in antibacterial
efficacy between herbal mouthwash and commercial productnin controling S. mutans
and L. acidophilus growth.

1.5 Methodology applied.

In experiment, focus is evaluating effectiveness of homemade herbal mouthwash

compared to commercially available products in managing bacterial growth in oral
cavity. methodology employed involved several key to ensure accurate assessment.
First, herbal extract was prepared using Soxhlet extractor. This process involved
extraction of active compounds from chosen herbs, resulting in concentrated solution.
Upon evaporation, extract attained murky, sticky consistency, indicative of presence of
bioactive ingredient.
to ensure appropriate dilution and solubility, herbal extract was diluted to various
concentration per miligram in 5% solution of dimethyl sulfoxide (DMSO). This step
aimed to create standardized concentration suitable for testing while maintaining
stability of herbal component.
Then, well diffusion method was used on MuellerHinton agar (MHA) plates. This
technique involved creation of well within agar medium, into which herbal extract
solutions, along with commercial mouthwash sample, as contrast. By observing zones
of inhibition around each well, susceptibility or resistance on antibacterial method to
herbal extract and commercial products which could be visualized and compared.
Throughout experiment, some control were meticulously employed to ensure
reliability of result. Positive and negative control were utilized to validate efficacy of
herbal extract and commercial mouthwashe against known strain of oral microorganism.
By employing methodological approach, experiment aim to give valuable perceptions
into potential of herbal mouthwash like effective alternative for controlling oral
microbiota, compared to conventional commercial products. Moreover, systemati
comparison with commercial mouthwashes allows for comprehensive assessment of
efficacy regarding oral hygiene practices and product choices.

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 1.6 Bacterial culture used

Streptococcus mutans is bacterium that causes tooth decay and dental caries by
producing acids and glucans from sucrose. Also, Streptococcus mutans is model
organism for studying biology, genetics, physiology, and of Gram-positive pathogens
that live in biofilms and interact with host immune system. its growth and activity in oral
cavity. (Lemos et al., 2019,).
According to Ahirwar, S. et al., 2021. Lactobacillus acidophilus is common
bacterium in oral problems that it may help us understand how this bacterium interacts
with other oral microbes and the host immune system. It may also help us develop new
strategies to prevent or treat oral diseases by modulating oral microbiome. L.
acidophilus is probiotic bacterium that can benefit oral cavity by producing lactic acid,
hydrogen peroxide, and bacteriocins.

1.7 Media to be used

Preparation of MHA (Mueller-Hinton agar) with components of Bacteriological

Agar powder VWR® brand and Mueller-Hinton broth powder by Himedia® brand. use of
this media provides standardized and reliable stage for assessing susceptibility of
bacterial strains like Streptococcus mutans and Lactobacillus acidophilus to herbal
extracts and commercial mouthwashes with evaluation of well diffusion method.
TSA (trypticase soy agar) plates already prepared by prep-room in Centennial
college lab. Also, it is used to preserve viability of bacteria during 7experiment, and for
maintenance purposes, it is necessary to keep sample cycle active throughout all
required assays.

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 METHODOLOGY

preparation of this experiment is conducted in laboratories HPSTC 452 and 446

at Centennial College, Morningside Campus, under supervision of Professor Natasha
Singh

2.1 Project Material list

2.1.2 Living Samples

The organisms to be used were supplied from culture stored in laboratories of
Centennial College, Morningside Campus: Streptococcus mutans ATCC#-25195, and
Lactobacillus acidophilus ATCC#-4356.

2.1.3 Herbal mouthwash components

materials to be used were sourced from regular Canadian chain of food or local
herbal medicinal alternatives. Pure samples were obtained, then dried and ground as
finely as possible using kitchen materials, before being transported to laboratory: loos
peppermint leaves (Mentha × piperita); Clove (Syzygium aromaticum); Cinnamon
(Cinnamomum verum).

2.1.4 Commercial Mouthwash Product

This mouthwash is formulated by Colgate® brand and carries Drug identification
number DIN-02443783 and LOT-2318U6561C. It contains cetylpyridinium chloride
(CPC) at concentration of 0.075% (w/w) antiseptic agent.

2.1.5 Chemicals and equipment to be used

 Distilled water
 Dimethyl sulfoxide (DMSO)
 Bacteriological Agar (powder)
 Mueller-Hilton broth (powder)
 Soxhlet extraction apparatus
 Boiling chips

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  cheesecloth
 Metalware set (support stand with clamps)
 TSA plates
 Empty plates
 PYREX® Petri Dishes with Cover (glass)
 Hot plate
 Beakers
 Erlenmeyer flasks
 Reagent bottles
 Media bottles
 Graduated cylinder.
 Bottom flask
 Stirring rod
 Aluminum foil
 Parafilm M film laboratory
 Pipettes.
 Micropipette
 Dry hood or desiccators.
 Spatula
 Test tubes and racks.
 Eppendorf tubes
 Weight balance
 Aseptic Swabs
 Tape and marker
 Logbook

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2.2 Experimental design:

2.2.1 Soxhlet extraction

This technique is used for extract herbal organic compounds from solid (powder
component) sample. three ingredients weighing total of 30 grams (well mixed) are used
and subjected to extraction process in Soxhlet apparatus. This mixture will be placed
while is Assembling Soxhlet Apparatus, Soxhlet apparatus consists of an extraction
flask, condenser, and receiving flask that is (bottom flask). mixture of ingredients is
placed in extraction flask, which is contained in porous container, such as cheesecloth,
to form "pellet" that remains inside apparatus during extraction process. Addition of
distilled Water: Approximately 300 ml of water is added to receiving flask (bottom flask).
Water acts as solvent for extracting compounds from sample.
Initiation of Extraction Cycle receiving flask (bottom flask) is heated, causing
water to evaporate and rise into condenser. condensed water vapor precipitates onto
sample in extraction flask and dissolves organic compounds present in solid sample
water containing dissolved compounds drips from condenser back into extraction flask,
where it extracts more compounds from sample. It is necessary to highligh input and
output systems of Soxhlet apparatus are connected to direct water supply of laboratory
to ensure continuous water supply throughout extraction process. This cycle continues
until compounds are concentrated enough in bottom flask, at which point Soxhlet
automatically returns solvent to extraction flask and begins new extraction cycle. The
Duration of Process can last several hours until multiple extraction cycles are
completed, and significant number of compounds is obtained in bottom flask discarding
boiling chips at end of this process.

2.2.2 Evaporation cycle

evaporation cycle is implemented throug process called "china dish evaporation."
This involves gradual heating, not exceeding 50 degrees Celsius in water bath, to avoid
burning or altering properties of extract while it dries, ensuring finer and purer
concentrate consistently. This process is conducted steadily and slowly, even allowing
residue to dry in Pyrex dishes or petri dishes placed in chambers or fume hoods at
slightly higher temperature than ambient. It is repeated over several days until desired
consistency or dryness is achieved in Pyrex dishes. Then, dried extract is mixed with
any remaining liquid extract to achieve appropriate consistency.

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2.2.3 Dilution of Concentrate:
In this case, study will be initiated to assess various concentrations for their
effectivenes upon contact with bacteria to be implemented on MHA plate. Five different
samples will be created: 100 mg/ml, 200 mg/ml, 300 mg/ml, 400 mg/ml, and 500 mg/ml.
This means weight measurement of milligrams of product (herbal extract evaporated)
will be diluted in 1 ml of 5% DMSO. Firstly, pure DMSO is diluted to 5%. For instance, 1
ml of pure DMSO is diluted in 20 ml of distilled water to give final solution of 5% DMSO
to used. For this procedure, Eppendorf tubes are used separately, and milligrams to be
used are weighed, adding 1 ml of 5% DMSO at end. mixture is then agitated and mixed
well using vortex, obtaining each diluted sample.

2.2.4 Preparation de MHA media:

Preparation of agar: agar preparation involves use of bacterial agar, MHB
(Mueller-Hinton broth), and powder; necessary quantity in grams is obtained for plates
to be used for all tests according to manufacturer's instructions, including internal or
negative controls, and well diffusion method test for zone of inhibition. Calculations are
made to use approximately 20 ml of agar in each empty plate. calculations assume QC
of 10%. agar solution is dispensed into containers, autoclaved for sterilization, and
allowed to cool before use. Once solidified, agar media is ready for culturing
microorganisms. Unused media can be stored in sterile containers at room temperature
or refrigerated. It's crucial to adhere to standard laboratory protocols and safety
measures throughout process.

2.2.5 Well diffusion method:

In well diffusion method conducted on Mueller-Hinton agar (MHA) plates,
systematic approach is employed to assess efficacy of various substances against oral
microbiota. procedure begins by inoculating MHA plates with standardized bacterial
cultures with swabbing method. Next, wells are carefully created in agar surface,
ensuring consistent spacing between each well. Subsequently, prepared herbal extract
dilutions, ranging from low to high concentrations, are added to separate wells, along
with additional control wells containing pure herbal extract, 5% DMSO solution, and
commercial mouthwash. Each substance is added to individual MHA plates to allow for
clear observation of any differences in inhibitory effects. It is very important that each
sample is carefully inoculated into wells using micropipette, approximately 120
microliters per well, ensuring consistent volume delivery in order to Follow incubation.
plates are incubated at 37 degrees Celsius to simulate human body temperature and
allow for optimal bacterial growth. Incubation periods vary, typically ranging from 24 to
48 hours, to assess effectiveness of substances against bacterial cultures. Therefore,
final step proceed to exam for zone of inhibition around each well, indicating
effectiveness of tested substances against bacterial culture. This methodological way
confirms thorough evaluation of antimicrobial propertie of herbal extract compared to

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control substances, facilitating identification of potential therapeutic agents for
controlling oral microbiota.

2.3 Control:

2.3.1 Positive control:

 Standardized (Mouthwash and herbal) concentration for assessing bacterial

susceptibility.
 Incubation of organism in MHA and TSA plate.

2.3.2 Negative control:

 Incubation od (5% DMSO) solution to account for any non-specific effects in MHA
plate.

2.3.3 Environmental control:

 Maintain consistent temperature incubation condition and using same bacterial
strain for consistency.

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 REFERENCE

Ahirwar, S. S., Ahirwar, A. K., Singh, S., & Singh, A. (2021). Distribution and molecular
characterisation of Lactobacilli in the oral cavity of children. Indian Journal of Dental
Research, Retrieved February 3, 2024, from
https://pubmed.ncbi.nlm.nih.gov/34269229/

Clove oil for toothache: Use and side effects. (2018, March 19), Retrieved February 25,
2024, from https://www.medicalnewstoday.com/articles/321256

Elementa Nano Silver Xylitol Dental Mints - Peppermint - 60ct. (n.d.). DentalStores.
Retrieved February 25, 2024, from
https://www.dentalstores.com/catalog/product_info.php?products_id=49401.

Lemos, J. A., Palmer, S. R., Zeng, L., Wen, Z. T., Kajfasz, J. K., Freires, I. A.,
Abranches, J., & Brady, L. J. (2019). The Biology of Streptococcus mutans.
Microbiology Spectrum, 7(1). https://doi.org/10.1128/microbiolspec.gpp3-0051-
2018

Yanakiev, S. (2020). Effects of Cinnamon (Cinnamomum spp.) in Dentistry: A

Review. Molecules, 25(18), 4184. https://doi.org/10.3390/molecules25184184

Kshirsagar, M. M., Dodamani, A. S., Karibasappa, G. N., Vishwakarma, P. K., Vathar, J.

B., Sonawane, K. R., Jadhav, H. C., & Khobragade, V. R. (2018). Antibacterial activity of
garlic extract on cariogenic bacteria: An in vitro study. Ayu, Retrieved February 3, 2024,
from The Effect of Various Oral Hygiene Products on Bacterial Growth - NASA/ADS
(harvard.edu)

Prasanth, M. (2011). Antimicrobial efficacy of different toothpastes and Mouthrinses: An

in vitro study. Dental research journal, Retrieved February 3, 2024, from Antimicrobial
Efficacy of Different Toothpastes and Mouthrinses: An In Vitro Study - PMC (nih.gov)

Kshirsagar, M. M., Dodamani, A. S., Karibasappa, G. N., Vishwakarma, P. K., Vathar, J.

B., Sonawane, K. R., Jadhav, H. C., & Khobragade, V. R. (2018). Antibacterial activity of
garlic extract on cariogenic bacteria: An in vitro study, Retrieved February 3, 2024, from
Antibacterial activity of garlic extract on cariogenic bacteria: An in vitro study - PMC
(nih.gov)

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