See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/282389537

Development of Alcohol-free Herbal Mouthwash Having Anticancer Property

Article · January 2013

CITATIONS READS

0 2,599

4 authors, including:

Souptik Bhattacharya Arnav Garai

Jadavpur University Saha Institute of Nuclear Physics
16 PUBLICATIONS   8 CITATIONS    6 PUBLICATIONS   8 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Bio remediation of used motor oil using indigenous microorganism isolated from automobile industry effluent View project

Development of herbal mouthcare produçts View project

All content following this page was uploaded by Arnav Garai on 03 October 2015.

The user has requested enhancement of the downloaded file.

 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

Abstract
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: ray.bit.70@gmail.com

INTRODUCTION
Various kinds of mouthwashes have evolved Another chemical alcohol free mouthwash
following oral hygienic problems. But apart consists of sodium chloride, sodium
from this, mouthwashes also serve to refresh bicarbonate, a flavoring agent and as a
breath. Moreover, mouthwash also contains solubilizing agent here for, polysorbate 20,
some ingredients that serve as digestive aids. being a mixture of partial lauric esters of
Mouthwashes can be chemical or herbal in sorbitol and its mono- and di-anhydrides
nature. copolymerized with approximately 20 mol of
ethylene oxide for each mol of sorbitol and its
Cetylpyridinium chloride (CPC) is a cationic anhydrides; and water, said sodium chloride
quaternary ammonium compound in some and sodium bicarbonate being dissolved in
types of mouthwashes, toothpastes, lozenges, said water and present in a suitable quantity to
throat sprays, anti-sore throat sprays, breath produce a solution substantially isotonic with
sprays, and nasal sprays. It is thus a type of human oral mucosa.
chemical mouthwash. It is an antiseptic that
kills bacteria and other microorganisms. It has A herbal guava mouthwash has been prepared
been shown to be effective in preventing using guava leaf extract, sodium lauryl
dental plaque and reducing gingivitis [1]. But sulphate, peppermint emulsion and chloroform
it has certain disadvantages. It is combustible. water. Formulations with highest quantity of
Concentrated solutions are destructive to guava leaf extract have highest antimicrobial
mucous membranes. It is toxic when activity due to the presence of bioactive
swallowed and very toxic when inhaled. 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 medium is now liquefied by keeping in water
development of new type of herbal mouthwash bath. Sterilized petridishes are kept in hot air
from guava leaves, peppermint oil, clove oil, oven for 5 min. Now, inside the laminar,
betel leaves and ajwain and to study briefly its 15 mL of liquefied agar is poured in
antibacterial properties and shelf life period. petridishes. The agar-filled petridishes are then
allowed to cool; 0.1 mL of mouthwash sample
MATERIALS AND METHODS is added to the pertidish maintaining sterilized
Test Organisms: A freshly prepared slant conditions. The mouthwash sample is then
culture of Streptococcus mutans MTCC-890 spread with a spreader. Then the petridishes
and E. Coli. are labeled and kept in the incubator at 37 °C
and then checked for appearance of colonies
Chemicals/Reagents Required: Culture after 24 h and after 7 days in the colony
media used were nutrient agar and nutrient counter.
broth. Sterile distilled water, alcohol (95%),
anthromycin, and edible apple green food Determination of Antibacterial Property of
color. Mouthwash
Twenty-four hour freshly prepared cultures of
Herbal Ingredients: Guava leaves, betel Streptococcus mutans and E. coli are used that
leaves, ajwain (local market), peppermint oil are prepared from slant cultures of
(local market), clove oil (local market). Streptococcus mutans and E. coli. This is done
by: nutrient broth is prepared, pH is tested and
Equipment Used: Dhona balance (160D, sterilized. Using a sterilized loop, some
Kolkata, India), autoclave (Instind, Kolkata, inoculum is transferred from old culture to the
India) hot air oven (Multispan MDC-1901), newly prepared nutrient broth in the laminar.
100 °C water bath (ICT, Kolkata, India ), These subcultures are now placed in
grinder, Whatman filter paper, laminar air flow incubators at 37 °C for 48 h. Petridishes are
colony counter, spreader, incubator (Lambda, filled with 15 mL agar media as above and
l/032/01-02-03), refrigerator (LG, Kolkata, allowed to get cooled. Using a marker, each
India). petridish is divided into two halves. 0.05 mL
of Streptococcus mutans is added to one
Preparation of Mouthwash: The distilled petridish and 0.05 mL of E. coli is added to the
water, glass wares are sterilized; the leaves are other. After adding the inoculum, the
weighed and washed in tap water. The leaves inoculum is spread by the spreader. The
are dried for 24 h in a hot air oven. The dried petridishes are kept in the incubator at 37 °C
leaves are now grinded in a grinder and the for 5 min. The petridishes are taken out and
powdered form is obtained. The leaf powder is placed in the laminar. Using the borer, wells
boiled in sterilized water for 15 min by are created in the inoculum-applied agar media
keeping it in the water bath (10 g/100 mL). on each of the halves of the petridishes. This
The liquid thus obtained is filtered using a method is known as the bore well method.
Whatman paper (40 mL of each extract). 0.05 mL of the refrigerated mouthwash sample
Clove oil and peppermint oil (each 2 mL) are is applied in one half and 0.05 mL of ambient
added finally to the extract mixture. The edible temperature mouthwash is placed in the other
food color is mixed in it. The sample thus half. This process is repeated respectively for
prepared is now kept in two volumetric flasks the Streptococcus mutans and E. coli
at two varied temperatures, one at ambient petridishes. The petridishes are now kept in
temperature and the other at low temperature. the incubator at 37 °C for 24 h. After 24 h,
petridishes are looked for inhibition zones
MICROBIAL ANALYSIS around the wells
Procedure for Shelf Life Determination
The shelf life of the sample was determined by Minimum Inhibitory Concentration of the
pour plate method with the diluted sample in Herbal Extract
water using total plate count method for viable This is the lowest concentration expressed in
cells. The agar medium is prepared, pH is mg/L that under given in vitro conditions
tested and autoclaved. The sterilized agar inhibit the growth of bacteria in a given period

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 herbal extract with the standard antibiotic. The
using a series of dilutions of the herbal sample diameter of the inhibition zone for both, the
in the liquid broth. The herbal sample is ten extract and antibiotic is measured. Hot agar is
times diluted from the original sample. After added to the petridishes in the laminar and
inoculation of the test strain in the test tubes, allowed to cool. Using the borer wells are
growth is determined by visible turbidity after made on the petridishes that have been divided
24 hr. MIC tests are also done by extended into two halves by the marker. The 500 mg
breakpoint sensitivity test and by using antibiotic is diluted to 250 ppm concentration
commercially available E-Test strips. These On the S side, 0.1 mL of the extract is added
methods are technically time consuming and and on the A side 0.1 mL of the antibiotic
expensive. The original herbal extract is althromycin (250 ppm) is added. The petridish
diluted ten times using 1 mL sample and 9 mL is kept in the incubator at 37 °C for 24 h. The
of sterile distilled water. The test tubes are diameters of the zone of inhibition are
filled with broth and the diluted herbal sample measured.
in varied proportions. The test tube containing
only broth is the positive control while the test Sensory Analysis
tube containing only herbal sample is the The color, flavor and taste of 0-day and 7th
negative control. day samples were determined using 9-point
Hedonic scale method.
0.1 mL of freshly prepared Streptococcus
mutans culture is added to all the test tubes Physiochemical Test
maintaining sterile conditions throughout. The The pH of the of 0-day and 7th day mouthwash
test tubes are now kept at incubator at 37 °C samples at ambient and low temperature
for 24 h and observed for the presence of storage are measured to monitor the changes
visible turbidity. 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

RESULTS
Table 1: Sensory Characteristics.
Sample Temperature Color Flavor Taste Texture
0 day Ambient 6 8 9 Clear
0 day Refrigerated 6 8 9 Clear
7 day Ambient 4 6.5 7 A little turbid
7 day Refrigerated 5 7 8 Clear

0-extrmely bad, 3-not bad not good, 5-moderate, 7-good, 9-excellent.

Color: The color change observed here is the Taste: The taste is strong and remains almost
original color of the mouthwash sample. The same over the week except for the ambient
color is dull brownish on the 0th day and temperature sample.
degrades faster at the ambient temperature.
Texture: Samples remain clear liquid over a
Flavor: The flavor is almost unchanged and week except the one kept at the ambient
has an excellent fragrance of clove and temperature which develops turbidity after the
peppermint. Only a week after, the fragrance is 3rd day.
somewhat lost when kept at ambient
temperature.

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 6.5
7th day 6.5

Thus, no change was found in the acidity of observed to be quite stable chemically.
the sample over a week and the sample was

Table 3: Microbial Analysis.

Storage Life (7 days)
Storage temperature of Storage life (log cfu/mL) – Storage life (log cfu/mL) –
sample 0th day seventh day
Ambient No growth 4.90
Refrigerated No growth 4.48

The sample at ambient temperature develops (Gram-positive bacteria) Streptococcus mutans

more colonies than the one at refrigerated – inhibition zone observed. So, antibacterial
temperature when observed over a week. property is observed against both
Streptococcus mutans (Gram-positive) and E.
Antibacterial Properties: E. coli (Gram- coli (Gram-negative).
negative bacteria) – Inhibition zone observed

MIC Method
Table 4: MIC

10D Extract Broth Dilution Result

0 5 Positive control +
0.5 0.5 0.1 (10D) +
1 4 0.2 (10D) +
1.5 3.5 0.3 (10D) -
2 3 0.4 (10D) -
2.5 2.5 0.5 (10D) -
3 2 0.6 (10D) -
3.5 1.5 0.7 (10D) -
4 1 0.8 (10D) -
5 0 Negative -
control
MIC was thus determined to be 3 mg/ml.

Agar Diffusion Method vitamin contents are calcium, carotene,

The diameter of inhibition zone for thiamine, riboflavin, niacin and vitamin C. Its
anthromycin – 30 + 0.5 mm. The diameter of calorific value is 44. Recent studies have
inhibition zone for original sample – shown that betel leaves contain tannins, sugar
20 + 0.5 mm. and diastases and an essential oil. The
Sample activity unit = 250 ppm essential oil is a light yellow liquid of aromatic
anthromycin × (20/30) unit = 166.66 anthro- odor and sharp burning in taste. It contains a
mycin unit. phenol called chavicol which has powerful
antiseptic properties. The alkaloid arakene in it
DISCUSSION has properties resembling cocaine in some
Betel Leaf: An analysis of the betel leaf respects. The betel leaf has analgesic and
shows it consists of moisture 85.4%, protein cooling properties. According to traditional
3.1%, fat 0.8%, minerals 2.3%, fiber 2.3% and Ayurveda medicine, chewing areca nut and
carbohydrates 6.1% per 100 g. Its minerals 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 with other species remaining unstudied. From
content, and is often used as tea and for preliminary medical research in laboratory
flavoring ice cream, confectionery, chewing models, extracts from apple guava leaves or
gum, and toothpaste. The oil also contains bark are implicated in therapeutic mechanisms
menthone and menthyl esters, particularly against cancer, bacterial infections,
menthyl acetate [3]. It is the oldest and most inflammation and pain [9–11]. Essential oils
popular flavor of mint-flavored confectionery. from guava leaves display anti-cancer activity
Peppermint can also be found in some in vitro [12]. Guava leaves are used in folk
shampoos and soaps, which give the hair a medicine as a remedy for diarrhea [13] and, as
minty scent and produce a cooling sensation well as the bark, for their supposed
on the skin [4]. Peppermint oil is versatile oil: antimicrobial properties and as an astringent.
it is analgesic, anti-inflammatory, anti-viral, Guava leaves or bark are used in traditional
digestive, anti-septic, an astringent, treatments against diabetes [14–16]. In
carminative and anti-spasmodic Peppermint, Trinidad, a tea made from young leaves is
essential oil is used to treat migraine, used for diarrhea, dysentery and fever [17],
bronchitis, sinusitis, indigestion, nausea, and against aging processes in human body.
irritable bowel syndrome, irregular periods Our skin cells contain fewer naturally
and nervous conditions. It is also very useful occurring antioxidants, as we age. It is helpful
in the treatment of cold and flu. in providing photo-protection, treating photo-
damaged skin and pigmentation disorders in
Clove: Cloves are used in Indian Ayurveda the skin. The antioxidant nature of guava
medicine, Chinese medicine, and Western enables it to reduce the harm caused to skin
herbalism and dentistry where the essential oil due to its exposure to sunlight. It serves as a
is used as an anodyne (painkiller) for dental mouth freshener. For people suffering from
emergencies. Cloves are used as a carminative diarrhea and amoebiasis, unripe guava is
to increase hydrochloric acid in the stomach useful for treating halitosis. It is rich in tannic,
and to improve peristalsis. Cloves are also said malic, oxalic and phosphoric acids as well as
to be a natural anthelmintic [5]. The essential calcium, oxalate and manganese. Chewing
oil is used in aromatherapy when stimulation fresh guava leaves also stops bleeding from
and warming are needed, especially for gums along with bad breath. Most
digestive problems. Topical application over mouthwashes contain antimicrobial substances
the stomach or abdomen are said to warm the as antiseptic ingredients against germs that
digestive tract. Clove oil, applied to a cavity in commonly cause mouth infections.
a decayed tooth, also relieves toothache [6]. Phytochemical studies indicate the guava
leaves bioactive components like tannins, tri
Ajwain: Ajwain is used as an antiseptic. It is terpenes, phenols, flavonoids, essential and
used for cleaning wounds and treating skin fixed oils, sapinins, lectins, carotenoids, etc.
infections. Thymol in Ajwain seeds is known However, despite the popular ehnomedical use
as a strong germicide, anti-spasmodic and of guava leaves there is still a dearth of
fungicide. Oil of Ajwain is also used in information on the use of guava leaves in the
toothpaste and perfumery. Ajwain leaves can mouthwash formulations. This study was
be crushed and applied on infections. Ajwain therefore carried out to determine the
seeds are also used in prevention of bad antimicrobial potency of guava leaves in
breath. Thymol from Ajwain seeds is also used mouthwash formulations. Along with all the
in various mouthwashes. Regularly chewing of use of individual constituents of the
Ajwain seeds along with fennel seeds prevents mouthwash, the herbs used are cheap, easy to
bad breath [7]. use and are easily available

Guava Leaves: Since the 1950s, guavas – Sample Sterility

particularly the leaves – have been the subject The herbal extract is prepared maintaining
for diverse research on their constituents, sterile conditions throughout and the sterility
pharmacological properties and history in folk is observed at two dilutions 10−1, 10−3.
medicine [8]. Most research, however, has No microbial growth was observed in the
been conducted on apple guava (P. guajava), sample prepared.

RRJoHS(2013) 7-12 © STM Journals 2013. All Rights Reserved Page 11

 Alcohol-free Herbal Mouthwash Having Anticancer Property Chowdhury et al.
__________________________________________________________________________________________

CONCLUSIONS 9. Chen Kuan-Chou, Hsieh et al. Brain

A sample of herbal mouthwash was developed derived metastatic prostate cancer DU-145
and tested for 0 and 7th day in ambient and cells are effectively inhibited in vitro by
refrigerated temperature and both are accepted guava (Psidium gujava L.) leaf extracts.
in respect to sensory and microbial Nutr. Cancer 2007 58(1): 93–106p.
characteristics. The developed sample has 10. Mahfuzul Hoque M.D., Bari M.L., Inatsu
positive effect on both S. mutans (Gram- Y., et al. Antibacterial activity of guava
positive) and E. coli (Gram-negative) culture. (Psidium guajava L.) and Neem
(Azadirachta indica A. Juss.) extracts
REFERENCES against foodborne pathogens and spoilage
1. Asadoorian Joanna. Cetylpyridinium bacteria. Foodborne Pathogens and
Chloride Mouth Rinse on Gingivitis And Disease 2007 4(4): 481–488p.
Plaque. Journal of Dental Hygiene 2008. 11. Manosroi J., Dhumtanom P., Manosroi A.
2. Naveen Pattnaik. The Tree of Life. PDR Anti-proliferative activity of essential oil
for Herbal Medicines, 4th Edn. Thomson extracted from Thai medicinal plants on
Healthcare: 640p. ISBN 978-1-56363- KB and P388 cell lines. Cancer Letters
678-3. 2006 235(1): 114–120p.
3. R. Eccles. Menthol and Related Cooling 12. Kaljee Linda M. Thiem, Vu Dinh, von
Compounds. J. Pharm. Pharmacol 1994; Seidlein, et al. Healthcare Use for
46(8):618–630p. PMID 7529306. Diarrhoea and Dysentery in Actual and
4. Balch Phyllis, Balch James. Prescription Hypothetical Cases, Nha Trang, Viet Nam.
for Nutritional Healing, 3rd edn. Avery Journal of Health, Population and
Publishing: 2000; 94p. Nutrition 2004; 22(2): 139–149p.
5. USA (2012-05-24). The Effect of Clove 13. Oh W.K., Lee C.H., Lee M.S., et al.
and Benzocaine versus Placebo as ... J Antidiabetic effects of extracts from
Dent 2006; PubMed-NCBI. Psidium guajava. J. Ethnopharmacol.
Ncbi.nlm.nih.gov. Retrieved 2012-09-07. 2005; 96(3): 411–415p.
6. http://valuefood.info/Herbs-and- 14. Mukhtar H.M., Ansari S.H., Bhat Z.A., et
Spices/Nutrition-Health-Benefits- al. Antidiabetic activity of an ethanol
Spices/health-benefits-of-ajowan-or- extract obtained from the stem bark of
ajwain-seeds.html Psidium guajava (Myrtaceae). Pharmazie
7. http://valuefood.info/Herbs-and- 2006; 61(8): 725–727.
Spices/Nutrition-Health-Benefits- 15. Y Deguchi, K Miyazaki. Anti-
Spices/health-benefits-of-ajowan-or- Hyperglycemic and Anti-Hyperlipidemic
ajwain-seeds.html Effects of Guava Leaf Extract, Medscape,
8. Ojewole J.A. Antiinflammatory and from Nutrition and Metabolism 2010.
analgesic effects of Psidium guajava Linn. 16. Mendes, John. Cote ce Cote la: Trinidad
(Myrtaceae) leaf aqueous extract in rats & Tobago Dictionary, Arima, Trinidad
and mice. Methods and Findings in 1986.
Experimental and Clinical Pharmacology 17. HI Abdelrahim, AZ Almagboul, MEA
2006; 28(7): 441–446p. Omer, et al. Antimicrobial Activity of
Psidium guava. L. Fitoterapia 2002;
73:713–715p.

RRJoHS (2013) 7-12 © STM Journals 2013. All Rights Reserved Page 12

View publication stats