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Herbal Mouthwash

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Development of Alcohol-free Herbal
Mouthwash Having Anticancer Property
Article · January 2013
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Arnav Garai
Saha Institute of Nuclear Physics
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Research & Reviews: Journal of Herbal Science
Volume 2, Issue 1, ISSN: 2278-2257
Development of Alcohol-free Herbal Mouthwash
Having Anticancer Property
Banani Ray Chowdhury*, Souptik Bhattacharya, Madhuparna Deb, Arnav Garai
Department of Biotechnology, Bengal Institute of Technology, West Bengal University of
Technology, India
Mouthwash is a liquid accessory to clean and maintain the health of our teeth for oral hygiene.
Nowadays, we use commercial mouthwash which contains many chemical compounds like sodium
lauryl sulfate, thymol, methyl salicylate, benzalkonium chloride, hydrogen peroxide, alcohol
which are harmful to our buccal cavity. We have developed a mouthwash with some common food
materials and herbs and which can replace costly chemicals like alcohol, coloring agents and
preservatives making our mouthwash economically more viable than commercial mouthwash. A
herbal mouthwash preparation is developed using the extracts of peppermint oil, clove oil, betel
leaf extract, ajwain, ginger, basil, etc., in sterilized conditions having antibacterial, anti-cancer,
anti-fungal activity. Basil leaves are known to reduce malodor and possess antibacterial property.
Ajwain helps in digestion and removal of malodour. The sensory, physiochemical and microbial
analysis of the mouthwash preparation was carried out on the day of preparation and on the
seventh day after sample preparation to determine its physiochemical changes, shelf life and its
antibacterial property (on the Gram-positive and Gram-negative bacteria) when the sample
prepared was kept under two different temperature conditions – ambient temperature and
refrigerated temperature.
Keywords: Oral hygiene, economically viable, antibacterial, malodour, shelf life
*Author for Correspondence E-mail: [email protected]
Various kinds of mouthwashes have evolved
following oral hygienic problems. But apart
from this, mouthwashes also serve to refresh
breath. Moreover, mouthwash also contains
some ingredients that serve as digestive aids.
Mouthwashes can be chemical or herbal in
Cetylpyridinium chloride (CPC) is a cationic
quaternary ammonium compound in some
types of mouthwashes, toothpastes, lozenges,
throat sprays, anti-sore throat sprays, breath
sprays, and nasal sprays. It is thus a type of
chemical mouthwash. It is an antiseptic that
kills bacteria and other microorganisms. It has
been shown to be effective in preventing
dental plaque and reducing gingivitis [1]. But
it has certain disadvantages. It is combustible.
Concentrated solutions are destructive to
mucous membranes. It is toxic when
swallowed and very toxic when inhaled.
Another chemical alcohol free mouthwash
consists of sodium chloride, sodium
bicarbonate, a flavoring agent and as a
solubilizing agent here for, polysorbate 20,
being a mixture of partial lauric esters of
sorbitol and its mono- and di-anhydrides
copolymerized with approximately 20 mol of
ethylene oxide for each mol of sorbitol and its
anhydrides; and water, said sodium chloride
and sodium bicarbonate being dissolved in
said water and present in a suitable quantity to
produce a solution substantially isotonic with
human oral mucosa.
A herbal guava mouthwash has been prepared
using guava leaf extract, sodium lauryl
sulphate, peppermint emulsion and chloroform
water. Formulations with highest quantity of
guava leaf extract have highest antimicrobial
activity due to the presence of bioactive
substances. It is more potent against
Streptococcus mutans and Escherichia coli.
RRJoHS(2013) 7-12 © STM Journals 2013. All Rights Reserved
Page 7
Alcohol-free Herbal Mouthwash Having Anticancer Property
Chowdhury et al.
So, our present project work is based on the
development of new type of herbal mouthwash
from guava leaves, peppermint oil, clove oil,
betel leaves and ajwain and to study briefly its
antibacterial properties and shelf life period.
Test Organisms: A freshly prepared slant
culture of Streptococcus mutans MTCC-890
and E. Coli.
Chemicals/Reagents Required: Culture
media used were nutrient agar and nutrient
broth. Sterile distilled water, alcohol (95%),
anthromycin, and edible apple green food
medium is now liquefied by keeping in water
bath. Sterilized petridishes are kept in hot air
oven for 5 min. Now, inside the laminar,
15 mL of liquefied agar is poured in
petridishes. The agar-filled petridishes are then
allowed to cool; 0.1 mL of mouthwash sample
is added to the pertidish maintaining sterilized
conditions. The mouthwash sample is then
spread with a spreader. Then the petridishes
are labeled and kept in the incubator at 37 °C
and then checked for appearance of colonies
after 24 h and after 7 days in the colony
Determination of Antibacterial Property of
Twenty-four hour freshly prepared cultures of
Streptococcus mutans and E. coli are used that
are prepared from slant cultures of
Streptococcus mutans and E. coli. This is done
by: nutrient broth is prepared, pH is tested and
sterilized. Using a sterilized loop, some
inoculum is transferred from old culture to the
newly prepared nutrient broth in the laminar.
These subcultures are now placed in
incubators at 37 °C for 48 h. Petridishes are
filled with 15 mL agar media as above and
allowed to get cooled. Using a marker, each
petridish is divided into two halves. 0.05 mL
of Streptococcus mutans is added to one
petridish and 0.05 mL of E. coli is added to the
other. After adding the inoculum, the
inoculum is spread by the spreader. The
petridishes are kept in the incubator at 37 °C
for 5 min. The petridishes are taken out and
placed in the laminar. Using the borer, wells
are created in the inoculum-applied agar media
on each of the halves of the petridishes. This
method is known as the bore well method.
0.05 mL of the refrigerated mouthwash sample
is applied in one half and 0.05 mL of ambient
temperature mouthwash is placed in the other
half. This process is repeated respectively for
the Streptococcus mutans and E. coli
petridishes. The petridishes are now kept in
the incubator at 37 °C for 24 h. After 24 h,
petridishes are looked for inhibition zones
around the wells
Procedure for Shelf Life Determination
The shelf life of the sample was determined by
pour plate method with the diluted sample in
water using total plate count method for viable
cells. The agar medium is prepared, pH is
tested and autoclaved. The sterilized agar
Minimum Inhibitory Concentration of the
Herbal Extract
This is the lowest concentration expressed in
mg/L that under given in vitro conditions
inhibit the growth of bacteria in a given period
Herbal Ingredients: Guava leaves, betel
leaves, ajwain (local market), peppermint oil
(local market), clove oil (local market).
Equipment Used: Dhona balance (160D,
Kolkata, India), autoclave (Instind, Kolkata,
India) hot air oven (Multispan MDC-1901),
100 °C water bath (ICT, Kolkata, India ),
grinder, Whatman filter paper, laminar air flow
colony counter, spreader, incubator (Lambda,
l/032/01-02-03), refrigerator (LG, Kolkata,
Preparation of Mouthwash: The distilled
water, glass wares are sterilized; the leaves are
weighed and washed in tap water. The leaves
are dried for 24 h in a hot air oven. The dried
leaves are now grinded in a grinder and the
powdered form is obtained. The leaf powder is
boiled in sterilized water for 15 min by
keeping it in the water bath (10 g/100 mL).
The liquid thus obtained is filtered using a
Whatman paper (40 mL of each extract).
Clove oil and peppermint oil (each 2 mL) are
added finally to the extract mixture. The edible
food color is mixed in it. The sample thus
prepared is now kept in two volumetric flasks
at two varied temperatures, one at ambient
temperature and the other at low temperature.
RRJoHS (2013) 7-12 © STM Journals 2013. All Rights Reserved
Page 8
Research & Reviews: Journal of Herbal Science
Volume 2, Issue 1, ISSN: 2278-2257
of time. Conventionally, this is determined
using a series of dilutions of the herbal sample
in the liquid broth. The herbal sample is ten
times diluted from the original sample. After
inoculation of the test strain in the test tubes,
growth is determined by visible turbidity after
24 hr. MIC tests are also done by extended
breakpoint sensitivity test and by using
commercially available E-Test strips. These
methods are technically time consuming and
expensive. The original herbal extract is
diluted ten times using 1 mL sample and 9 mL
of sterile distilled water. The test tubes are
filled with broth and the diluted herbal sample
in varied proportions. The test tube containing
only broth is the positive control while the test
tube containing only herbal sample is the
negative control.
0.1 mL of freshly prepared Streptococcus
mutans culture is added to all the test tubes
maintaining sterile conditions throughout. The
test tubes are now kept at incubator at 37 °C
for 24 h and observed for the presence of
visible turbidity.
herbal extract with the standard antibiotic. The
diameter of the inhibition zone for both, the
extract and antibiotic is measured. Hot agar is
added to the petridishes in the laminar and
allowed to cool. Using the borer wells are
made on the petridishes that have been divided
into two halves by the marker. The 500 mg
antibiotic is diluted to 250 ppm concentration
On the S side, 0.1 mL of the extract is added
and on the A side 0.1 mL of the antibiotic
althromycin (250 ppm) is added. The petridish
is kept in the incubator at 37 °C for 24 h. The
diameters of the zone of inhibition are
Sensory Analysis
The color, flavor and taste of 0-day and 7th
day samples were determined using 9-point
Hedonic scale method.
Physiochemical Test
The pH of the of 0-day and 7th day mouthwash
samples at ambient and low temperature
storage are measured to monitor the changes
of acidity of the sample, considering pH < 7 to
be suitable for our mouth.
Standardization by Agar Diffusion Method
This is done to compare the strength of the
0 day
0 day
7 day
7 day
Table 1: Sensory Characteristics.
Temperature Color
A little turbid
0-extrmely bad, 3-not bad not good, 5-moderate, 7-good, 9-excellent.
Color: The color change observed here is the
original color of the mouthwash sample. The
color is dull brownish on the 0th day and
degrades faster at the ambient temperature.
Flavor: The flavor is almost unchanged and
has an excellent fragrance of clove and
peppermint. Only a week after, the fragrance is
somewhat lost when kept at ambient
Taste: The taste is strong and remains almost
same over the week except for the ambient
temperature sample.
Texture: Samples remain clear liquid over a
week except the one kept at the ambient
temperature which develops turbidity after the
3rd day.
RRJoHS(2013) 7-12 © STM Journals 2013. All Rights Reserved
Page 9
Alcohol-free Herbal Mouthwash Having Anticancer Property
Chowdhury et al.
Table 2: Physiochemical Characteristics.
Day of Measurement
pH of the Sample
0th day
7th day
Thus, no change was found in the acidity of
the sample over a week and the sample was
observed to be quite stable chemically.
Table 3: Microbial Analysis.
Storage Life (7 days)
Storage temperature
Storage life (log cfu/mL) –
0th day
No growth
No growth
The sample at ambient temperature develops
more colonies than the one at refrigerated
temperature when observed over a week.
Antibacterial Properties: E. coli (Gramnegative bacteria) – Inhibition zone observed
Storage life (log cfu/mL) –
seventh day
(Gram-positive bacteria) Streptococcus mutans
– inhibition zone observed. So, antibacterial
Streptococcus mutans (Gram-positive) and E.
coli (Gram-negative).
MIC Method
Table 4: MIC
10D Extract
Positive control
0.1 (10D)
0.2 (10D)
0.3 (10D)
0.4 (10D)
0.5 (10D)
0.6 (10D)
0.7 (10D)
0.8 (10D)
MIC was thus determined to be 3 mg/ml.
Agar Diffusion Method
The diameter of inhibition zone for
anthromycin – 30 + 0.5 mm. The diameter of
inhibition zone for original sample –
20 + 0.5 mm.
Sample activity unit = 250 ppm
anthromycin × (20/30) unit = 166.66 anthromycin unit.
Betel Leaf: An analysis of the betel leaf
shows it consists of moisture 85.4%, protein
3.1%, fat 0.8%, minerals 2.3%, fiber 2.3% and
carbohydrates 6.1% per 100 g. Its minerals and
vitamin contents are calcium, carotene,
thiamine, riboflavin, niacin and vitamin C. Its
calorific value is 44. Recent studies have
shown that betel leaves contain tannins, sugar
and diastases and an essential oil. The
essential oil is a light yellow liquid of aromatic
odor and sharp burning in taste. It contains a
phenol called chavicol which has powerful
antiseptic properties. The alkaloid arakene in it
has properties resembling cocaine in some
respects. The betel leaf has analgesic and
cooling properties. According to traditional
Ayurveda medicine, chewing areca nut and
betel leaf is a remedy for bad breath [2].
RRJoHS (2013) 7-12 © STM Journals 2013. All Rights Reserved
Page 10
Research & Reviews: Journal of Herbal Science
Volume 2, Issue 1, ISSN: 2278-2257
Peppermint: Peppermint has a high menthol
content, and is often used as tea and for
flavoring ice cream, confectionery, chewing
gum, and toothpaste. The oil also contains
menthone and menthyl esters, particularly
menthyl acetate [3]. It is the oldest and most
popular flavor of mint-flavored confectionery.
Peppermint can also be found in some
shampoos and soaps, which give the hair a
minty scent and produce a cooling sensation
on the skin [4]. Peppermint oil is versatile oil:
it is analgesic, anti-inflammatory, anti-viral,
carminative and anti-spasmodic Peppermint,
essential oil is used to treat migraine,
bronchitis, sinusitis, indigestion, nausea,
irritable bowel syndrome, irregular periods
and nervous conditions. It is also very useful
in the treatment of cold and flu.
Ajwain: Ajwain is used as an antiseptic. It is
used for cleaning wounds and treating skin
infections. Thymol in Ajwain seeds is known
as a strong germicide, anti-spasmodic and
fungicide. Oil of Ajwain is also used in
toothpaste and perfumery. Ajwain leaves can
be crushed and applied on infections. Ajwain
seeds are also used in prevention of bad
breath. Thymol from Ajwain seeds is also used
in various mouthwashes. Regularly chewing of
Ajwain seeds along with fennel seeds prevents
bad breath [7].
with other species remaining unstudied. From
preliminary medical research in laboratory
models, extracts from apple guava leaves or
bark are implicated in therapeutic mechanisms
inflammation and pain [9–11]. Essential oils
from guava leaves display anti-cancer activity
in vitro [12]. Guava leaves are used in folk
medicine as a remedy for diarrhea [13] and, as
well as the bark, for their supposed
antimicrobial properties and as an astringent.
Guava leaves or bark are used in traditional
treatments against diabetes [14–16]. In
Trinidad, a tea made from young leaves is
used for diarrhea, dysentery and fever [17],
and against aging processes in human body.
Our skin cells contain fewer naturally
occurring antioxidants, as we age. It is helpful
in providing photo-protection, treating photodamaged skin and pigmentation disorders in
the skin. The antioxidant nature of guava
enables it to reduce the harm caused to skin
due to its exposure to sunlight. It serves as a
mouth freshener. For people suffering from
diarrhea and amoebiasis, unripe guava is
useful for treating halitosis. It is rich in tannic,
malic, oxalic and phosphoric acids as well as
calcium, oxalate and manganese. Chewing
fresh guava leaves also stops bleeding from
gums along with bad breath. Most
mouthwashes contain antimicrobial substances
as antiseptic ingredients against germs that
Phytochemical studies indicate the guava
leaves bioactive components like tannins, tri
terpenes, phenols, flavonoids, essential and
fixed oils, sapinins, lectins, carotenoids, etc.
However, despite the popular ehnomedical use
of guava leaves there is still a dearth of
information on the use of guava leaves in the
mouthwash formulations. This study was
therefore carried out to determine the
antimicrobial potency of guava leaves in
mouthwash formulations. Along with all the
use of individual constituents of the
mouthwash, the herbs used are cheap, easy to
use and are easily available
Guava Leaves: Since the 1950s, guavas –
particularly the leaves – have been the subject
for diverse research on their constituents,
pharmacological properties and history in folk
medicine [8]. Most research, however, has
been conducted on apple guava (P. guajava),
Sample Sterility
The herbal extract is prepared maintaining
sterile conditions throughout and the sterility
is observed at two dilutions 10−1, 10−3.
No microbial growth was observed in the
sample prepared.
Clove: Cloves are used in Indian Ayurveda
medicine, Chinese medicine, and Western
herbalism and dentistry where the essential oil
is used as an anodyne (painkiller) for dental
emergencies. Cloves are used as a carminative
to increase hydrochloric acid in the stomach
and to improve peristalsis. Cloves are also said
to be a natural anthelmintic [5]. The essential
oil is used in aromatherapy when stimulation
and warming are needed, especially for
digestive problems. Topical application over
the stomach or abdomen are said to warm the
digestive tract. Clove oil, applied to a cavity in
a decayed tooth, also relieves toothache [6].
RRJoHS(2013) 7-12 © STM Journals 2013. All Rights Reserved
Page 11
Alcohol-free Herbal Mouthwash Having Anticancer Property
Chowdhury et al.
A sample of herbal mouthwash was developed
and tested for 0 and 7th day in ambient and
refrigerated temperature and both are accepted
in respect to sensory and microbial
characteristics. The developed sample has
positive effect on both S. mutans (Grampositive) and E. coli (Gram-negative) culture.
1. Asadoorian Joanna. Cetylpyridinium
Chloride Mouth Rinse on Gingivitis And
Plaque. Journal of Dental Hygiene 2008.
2. Naveen Pattnaik. The Tree of Life. PDR
for Herbal Medicines, 4th Edn. Thomson
Healthcare: 640p. ISBN 978-1-56363678-3.
3. R. Eccles. Menthol and Related Cooling
Compounds. J. Pharm. Pharmacol 1994;
46(8):618–630p. PMID 7529306.
4. Balch Phyllis, Balch James. Prescription
for Nutritional Healing, 3rd edn. Avery
Publishing: 2000; 94p.
5. USA (2012-05-24). The Effect of Clove
and Benzocaine versus Placebo as ... J
Ncbi.nlm.nih.gov. Retrieved 2012-09-07.
6. http://valuefood.info/Herbs-andSpices/Nutrition-Health-BenefitsSpices/health-benefits-of-ajowan-orajwain-seeds.html
7. http://valuefood.info/Herbs-andSpices/Nutrition-Health-BenefitsSpices/health-benefits-of-ajowan-orajwain-seeds.html
8. Ojewole J.A. Antiinflammatory and
analgesic effects of Psidium guajava Linn.
(Myrtaceae) leaf aqueous extract in rats
and mice. Methods and Findings in
Experimental and Clinical Pharmacology
2006; 28(7): 441–446p.
9. Chen Kuan-Chou, Hsieh et al. Brain
derived metastatic prostate cancer DU-145
cells are effectively inhibited in vitro by
guava (Psidium gujava L.) leaf extracts.
Nutr. Cancer 2007 58(1): 93–106p.
10. Mahfuzul Hoque M.D., Bari M.L., Inatsu
Y., et al. Antibacterial activity of guava
(Psidium guajava L.) and Neem
(Azadirachta indica A. Juss.) extracts
against foodborne pathogens and spoilage
bacteria. Foodborne Pathogens and
Disease 2007 4(4): 481–488p.
11. Manosroi J., Dhumtanom P., Manosroi A.
Anti-proliferative activity of essential oil
extracted from Thai medicinal plants on
KB and P388 cell lines. Cancer Letters
2006 235(1): 114–120p.
12. Kaljee Linda M. Thiem, Vu Dinh, von
Seidlein, et al. Healthcare Use for
Diarrhoea and Dysentery in Actual and
Hypothetical Cases, Nha Trang, Viet Nam.
Journal of Health, Population and
Nutrition 2004; 22(2): 139–149p.
13. Oh W.K., Lee C.H., Lee M.S., et al.
Antidiabetic effects of extracts from
Psidium guajava. J. Ethnopharmacol.
2005; 96(3): 411–415p.
14. Mukhtar H.M., Ansari S.H., Bhat Z.A., et
al. Antidiabetic activity of an ethanol
extract obtained from the stem bark of
Psidium guajava (Myrtaceae). Pharmazie
2006; 61(8): 725–727.
15. Y
AntiHyperglycemic and Anti-Hyperlipidemic
Effects of Guava Leaf Extract, Medscape,
from Nutrition and Metabolism 2010.
16. Mendes, John. Cote ce Cote la: Trinidad
& Tobago Dictionary, Arima, Trinidad
17. HI Abdelrahim, AZ Almagboul, MEA
Omer, et al. Antimicrobial Activity of
Psidium guava. L. Fitoterapia 2002;
RRJoHS (2013) 7-12 © STM Journals 2013. All Rights Reserved
Page 12
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