Article 5

International Journal of Pharmaceutical Research and Applications
Volume 7, Issue 3 May-June 2022, pp: 720-728 www.ijprajournal.com ISSN: 2456-4494
Preparation and Evaluation of Herbal Mouthwash

Abhishek D. Purohit*, Manisha U. Mishra, Vithika V. Gupta,Ayush K.
Kshirsagar , Akash S. Baghele And Yamini P. Pardhi.
Manoharbhai Patel Institute ofBachelor of Pharmacy,KudwaGondia, M.S.-441614, India.
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Submitted: 15-05-2022 Revised: 20-05-2022 Accepted: 25-05-2022
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ABSTRACT: Medicinal plant or herbs are as for their analgesic, anti-inflammatory or anti-
considered to have rich source of ingredients which fungal action. Additionally, some rinses act as
can be used in drug development. They can prevent saliva substitutes to neutralize acid and keep the
and cure disease because of their antimicrobial and mouth moist in xerostomia (dry mouth).Cosmetic
antibacterial property against microorganisms. The mouth rinses temporarily control or reduce bad
main objective of this literature work is to prepare breath and leave the mouth with a pleasant taste.
and evaluate a herbal mouthwash and check its Herbal Mouthwashes are in high demand,
effectiveness against microorganisms of oral because they act on oral pathogens and relieve the
cavity. To prepare Antibacterial herbal Mouthwash pain instantly and are also less side-effective.
from aqueous extract of 7 different Herbs namely Chemical mouthwashes have hydrogen peroxide
Azadirachtaindica (Neem), Ocimum and chlorhexidine as an immediate whitener,
bacilicum(Tulsi), Menthalogifolia(Mint), Curcuma sterilizer and pain reliever of teeth, but they tend to
longa(Turmeric), Syzygiumaromaticum(Clove), produce discoloration of teeth and may produce
Glycyrrhizaglabra(Liquorice), side effect, meanwhile they are cost effective. One
Cinnamomumverum(Cinnamon). That act against of the most common infectious diseases
the oral Staphylococcus aureus,E.coli and to check encountered by many individuals is Dental carries
the antimicrobial activity by using Agar well and Periodontal diseases at different stages of their
diffusion method. The medicinal plants which were life time.
suitable were collected and their water soluble The mouth washes are concentrated
extracts was prepared. The prepared mouthwash aqueous anti-bacterial solution that are used against
was evaluated against pathogens and was found oral microbes to counter oral infection, cleansing,
effective for controlling and demolishing microbial to get rid of bad breath refreshing ,anti-septic .The
growth in mouth. Further physiochemical mouthwash plats an prominent role in the oral
properties were tested for prepared mouthwash and hygiene of an individual ,it helps to relieve
itpossesses a good antibacterial activity. The symptoms of inflamed gums gingivitis. And also it
current mouthwash is liquid formulation with reliably used to destruct the pathogenic germs. The
potent action. mouth washes are used by most of the dental
patients to overcome sour mouth (xerostomia),
KEYWORDS: Herbal mouthwash, Natural ulcerated throat and sensitive teeth. Dentists always
extracts, Plaque maintenance, Antimicrobial, use mouthwash as an antimicrobial agent before
Staphylococcus aureus, and E.coli. oral surgery of the patients, because they help to
sterilize the surface of the inflamed gums and teeth,
I. INTRODUCTION: thereby the contamination of any other
Mouthwash, mouth rinse, oral rinse, or microorganisms can be avoided.
mouth bath is a liquid which is held in the mouth The uses of Neem are known from ancient
passively or swilled around the mouth by dates back since 45,000 years against oral diseases.
contraction of the perioral muscles and/or The neem leaves Azadirachtaindica belongs to the
movement of the head, and may be gargled, where family Lamiaceae. The neem solutions are used in
the head is tilted back and the liquid bubbled at the decreasing the inflammation of gums, to remove
back of the mouth. Usually mouthwashes are plaque and against dental cavities. Nowadays the
antiseptic solutions intended to reduce the neem extracts are used as antiseptic substance
microbial load in the mouth, although other against inflammation of mouth. The neem extract
mouthwashes might be given for other reasons such shows significant effects on both Gram positive
DOI: 10.35629/7781-0703720728 | Impact Factor value 7.429 | ISO 9001: 2008 Certified Journal Page 720
and Gram negative bacteria such as Staphylococcus carvacrol (3 percent) and eugenol-methyl-ether (20
aureus, E.coli. The inhibition is against the percent). It also contains caryophyllin in seeds
production of insoluble glucan,which is the main which contain fixed oil with good dental properties.
cause for plaque formation. Theneem sticks has The plant is also reported to contain alkaloids,
proven to show markedly inhibition against glycosides, saponin, tannins, an appreciable
Streptococci ,whichcolonize the surface of the amount of vitamin C and traces of maleic, citric
tooth. and tartaric acid.
Turmeric (Curcuma longa) a rhizomatous, Cloves((Syzygiumaromaticum)belongs to
herbaceous plant of the ginger family family Myrtaceaehave been shown to have
zingiberaceae. It is native to south west India antimicrobial properties, meaning they can help
requiring temperatures between 20º and 30º C and stop the growth of microorganisms like bacteria
the plants are gathered annually for their rhizomes. One test-tube study showed that clove essential oil
Turmeric was first used as a dye by ancients, then killed three common types of bacteria, including E.
later for its medicinal properties. The important coli, which is a strain of bacteria that can cause
chemical components of turmeric are a group of food poisoning.What’s more, the antibacterial
compounds called curcuminoids that includes properties of cloves could even help promote oral
demethoxycurcumin and bisdemethoxycurcumin. health .In one test-tube study, the compounds
The other volatile compounds include turmerone, extracted from cloves were found to stop the
altantone and zingiberine. According to the growth of two types of bacteria that contribute to
National centre for complementary and Integrative gum disease. Another study in 40 people tested the
health, some researches shows compound in effects of an herbal mouthwash consisting of tea
turmeric to have antifungal and antibacterial tree oil, cloves, and basil .After using the herbal
properties. mouthwash for 21 days, they showed
The Mentha leaves (Menthalongifolia) are improvements in gum health, as well as the amount
aromatic, perennial herbs. The menthe belongs to of plaque and bacteria in the mouth .In combination
the family Lamiaceae. Peppermint was first with regular brushing and proper oral hygiene, the
described in 1753 by Carl Linnaeus. Mentha are antibacterial effects of cloves may benefit your oral
extensively used as flavouring ingredient in breath health.Clove contains volatile oil, about 14-20 per
freshness, antiseptic mouth rinses, chewing gums cent and gallotannic acid, 10-13 per cent. Clove oil
and tooth paste. The pulegone primarily contains about 84-95 per cent of eugenol, about 3
responsible for the aroma and flavour of spearmint per cent of eugenol acetate and 5-8 per cent ß-
is L-Carvone. Menthol oil contains menthone, caryophyllene. Other constituents which are
menthyl esters, menthyl acetate and menthofuran. present in trace quantities include esters, ketones
Mint also consists of small amounts of many and alcohols. Clove oil has been reported to contain
additional compounds including Limonene, some 28 constituents and is devoid of
Pulegone, Caryophyllene and Pinene. monoterpenoids.
Tulsi(Ocimum bacilicum) is a legendary Liquorice(Glycyrrhizaglabra) belongs to
herb which has been used for ages due to its family FabaceaeThe root has long been used in
religious and medicinal values. Several Chinese and alternative medicine to enhance the
pharmacological studies have established a efficacy of other herbal remedies. More recently,
scientific basis for therapeutic uses of this plant studies have discovered that two other compounds,
belongs to family Lamiaceae. . It can prove licoricidin and licorisoflavan-A, found in dried
beneficial in treating oral diseases also because of licorice root, act as an effective antibacterial agent
its antibacterial, anti-inflammatory, ulcer healing, that can prevent or reduce the growth of bacteria
antioxidant, immunomodulatory properties. Future connected with tooth decay and gum disease.The
studies should be directed to explore and evaluate major constituent of the roots of G glabra and G
therapeutic significance of this miraculous plant in uralensis is a sweet triterpenesaponin glycyrrhizin,
periodontal diseases.Tulsi leaves contain bright, which is a potassium and calcium salt of
yellow coloured and pleasant volatile (0.1 - 0.9 per glycyrrhizic acid in the range of 6-14 per cent. It is
cent). The oil content of the drug varies depending a triterpene of oleanan skeleton, which after
up the type, the place of cultivation and season of hydrolysis, affords two molecules of glucuronic
its collection. The oil is collected by steam acid and an aglycone, glycyrrhetinic acid.
distillation method from the leaves and flowering Cinnamon(Cinnamomumverum) belongs
tops. It contains approximately 70 per cent eugenol, to family Lauraceae.The main reservoir for
Candida in the oral cavity has found to be the II. MATERIALS AND METHODS:
buccal mucosa and tongue. It has also been found Collection of Plant Leaves:Leaves of
to co-aggregate with bacteria in the sub gingival Azadirachtaindica(Neem),
biofilm and adhere to the epithelial cells and invade Ocimumteniflorum(Tulsi), Mentha (Mint), bark of
the gingival connective tissue. It is also found in Cinnamomumverum (cinnamon), flower bud of
the periodontal pocket of chronic periodontitis syzygiumaromaticum (clove), stem of
patients. Cinnamon being an ancient spice in the Glycyrrhizaglabara ( Liquorice) and rhizomes of
kitchen has been found to have multiple medicinal Curcuma longa (Turmeric) were randomly
values too. Cinnamomumzeylanicum and collected from mature plants.
Cinnamom cassia are the only two approved Extraction process:The leaves,stem and bark were
medicinal herbs of the genus Cinnamomum. It has washed with sterile water, shadow dried,
been found to have anti-cholesterol, anti-bacterial pulverized and stored in air-tight bottles. The
as well as anti-fungal property. Cinnamon in Aqueous extracts were prepared by soaking the
addition to its popularity as a spice has been found powdered leaves, stem and bark in sterile distilled
to have medicinal properties such as anti-bacterial, water and maintained in Incubator at 37±2°C for 72
anti-fungal, anti-oxidant.Cinnamon oil contains 60 hours and were filtered using Whattmann filter
- 70 per cent of cinnamaldehyde, 5 - 10 per cent paper.
eugenol, benzaldehyde, cuminaldehyde and other Equipments: Sterile Petri plates, volumetric flasks,
terpenes like phellandrene, pinene, cymene, Test tubes, Conicalflask, Whattmann filterpaper,
caryophyllene, etc. mortar pestle, Incubator, Autoclave, Pippetting
The aim to prepare Anti-bacterial Herbal device, Hot air-oven.
Mouthwash from the aqueous extracts of 7 Formulation of Herbal Mouthwash:Different
different herbs namelyAzadirachtaindica (Neem), mouthwashes containing various herbs were
Ocimum bacilicum(Tulsi), Menthalogifolia(Mint), prepared using established standard procedures.
Curcuma longa(Turmeric), The selection of the herbs were made, keeping in
Syzygiumaromaticum(Clove), mind the anti-microbial efficacy along with their
Glycyrrhizaglabra(Liquorice), excipients properties namely, preservative,
Cinnamomumverum(Cinnamon)that acts against sweetening and flavoring effects, which are
the oral pathogens- Staphylococcus aureus and required to develop an ideal mouthwash.Weighed
Escherchia coli to check the Anti-microbial quantity of each ingredient will be taken Extract
activity by using Agar well diffusion method. were taken mixed thoroughly in mortar and pestle
AIM:To perform the agar well diffusion technique property with small quantity of water. All other
in the Muller Hinton agar and to identify the anti- remaining ingredient will be gradually added with
bacterial activity of leaf extracts against test good mixing. Finally, water added to make volume
organisms, by measuring the zone of inhibition. and preservative ( alcohol 70%) will be added and
OBJECTIVE:To compare the efficacy of extracts the product will be packed in an ambered coloured,
of 7 different herbs on the basis of their resistance well closed container. The herbal Mouthwash was
and sensitivity towards the oral microbes and to prepared by the formula given in table.
formulate a herbalmouthwash.
TABLE 1:COMPOSITION OF HERBAL MOUTHWASH

Ingredient
Sr.no Botanicalname Role Quantity
s
1 Turmeric Curcuma longa Antibacterial 1ml
Azadirachta
2 Neem Antimicrobial 2ml
Indica
3 Tulsi Ocimumtenuiflorum Dental care 4ml
Anti-microbial,
4 Mint Mentha 2ml
Flavoring agent
5 Clove Syzygiumaromaticum Anti-inflammatory 1.5 ml
6 Cinnamon Cinnamomumverum Sweetening agent 2.5ml
7 Liquorice Glycyrrhizaglabara Anti-bacterial 2 ml
Aqueous extracts of Leaves by Shadow drying mouthwash was weighed and dissolved in 50 ml of
technique: distilled water and its pH was measured by pH
1. The leaves of mature plants were collected and meter.
washed 3 to 5 times with tap water to remove dust Test for microbial growth in formulated
and dirt. mouthwash:The formulated mouthwash was
2. The leaves were allowed to soak in already inoculated in the plates of agar media by streak
boiled water bath at 30-40°C for 10 to 15 minutes plate method and a control was prepared. The
to kill microbes present in the surface of the leaves.plates were placed in the incubator and are
3. The leaves undergoes Shadow drying technique incubated at 37±2°C for 24 hours. After the
were the leaves were spread in sterile container incubation period, the plates were taken out and
trays and kept at ambient temperature for 5 days. checked for the microbial growth by comparing it
4. After 5 days, the dried leaves were taken and with the control or standard.
powdered by using sterile mixer under aseptic Stability Studies:The formulation and preparation
condition. of any product is incomplete without proper
5. The pulverized leaves are transferred to air-tight stability studies of the prepared product. A general
sterile container jars. method for predicting the stability of any product is
6. 100ml of sterile distilled water was taken in accelerated stability studies, where the product is
conical flasks (250 ml), the pulverized leaves were subjected to elevated temperatures as per the ICH
weighed and suspended in distilled water under guidelines. A short term accelerated stability study
sterile condition. was carried out for the period of 1 month for the
7. The preparation was heat sterilized at 40°C for prepared formulation. The samples were stored
5-10 minutes and was kept for incubation at under the following conditions of temperature as 3-
37±2°C for 72 hours. 50° C, 250 C RH-60%, 400 C±2% RH- 75%.
8. After incubation, the extracts were filtered with Finally the samples were kept under accelerated
the help of a sterile Whattmann filter paper no: 1 studies and were withdrawn on weekly intervals
and a funnel under lab condition. and were analyzed.
9. The filtered extracts are boiled vigorously again In-vitro antibacterial activity: In-vitro
to kill the bacterial spores, which will prevent from antibacterial activity was performed on isolated
contamination. colonies of Streptococcus aureus. The Agar well
10. The extracts after heating is ready to use for thediffusion technique was used for determining the
formulation of Mouth wash and also can be tested zone of inhibition. The strains of S. aureuswere
against the oral pathogens by using Agar well inoculated in prefabricated agar plate. Plates were
diffusion techniques. dried and 4 wells were made with the help of 6 mm
agar well cutter. 20μl, 40μl, 60μl, 80μl of prepared
Evaluation Of Prepared Mouthwash mouthwash was loaded in all the respective wells.
Color and Odour:Physical parameters like odour The agar plates were kept undisturbed to allow the
and colour were tested by visual examination. passive diffusion of herbal mouth wash into the
pH:pH of prepared herbal mouthwash was agar culture medium. Then the plates were
measured by using digital pH meter. The pH meter incubated at 37±2°C for 24 hours. The zone of
was calibrated using standard buffer about 1 ml of inhibition was calculated in mm.
TABLE 2: CHEMICAL TEST
Sr No Name of herbs Tests Observation
1 Neem Aq. Extract + ferric chloride 5% Dark colourisation

solution
2 Tulsi 5 mg extract of Ocimum sanctum in Red colour

test tube + 1% HCl
3 Clove Aq. Extract + ferric chloride 5% Dark colourisation
solution
4 Turmeric Take turmeric powder in transparent Pink Colour
glass + few drop of water and
conc.HCl + shake it vigorously
5 Cinnamon Aq. Extract + potassium Decolourisation
permanganate
6 Liquorice Aq. Extract + lead acetate reagent White precipitate
7 Mint 5 mg extract of Ocimum sanctum in Red colour

test tube + 1% HCl
III. RESULTS AND DISCUSSION: combined with daily brushing and flossing.
The pH of the formulation was found to be Volatile sulfur compounds are the major
6.1. As the skin is having an acidic pH around 5.5 contributing factor to bad oral odour. They arise
this pH range of the formulation is suitable for oral from a variety of sources that is breakdown of
disorders. The formulation was found to be free food, dental plaque and bacteria associated with
from heavy metals. The formulation was free from oral disease.
microbes as they have not produced any microbial The antibacterial activity was evaluated by
growth when they got inoculated in the agar agar diffusion method for different concentrations
medium. This mouthwash is a purely herbal of mouthwash. The result of one of inhibition for
prepared without the addition of any kind of S.aureus was found to be 18 mm for 80μl, 15 mm
additives as other products found in the market. for 60μl, 12 mm for 40μl and 7 mm for 20μl
The formulation was undertaken stability studies respectively. These results showed that the herbal
for physical and chemical change. No considerable mouthwash has significant antibacterial activity
variations in properties of the formulation were and the present preparation is able to inhibit
observed. The results of stability studies are shown bacterial growth in oral cavity. The association of
in the given table.When used in mouthwashes oral microbial load on oral diseases in well
antimicrobial ingredient neem, tulsi, liquorice, established, thus this herbal mouthwash helps in
cinnamon, mint turmeric and clove extracts have motivating good oral health.
been found to reduce plaque and gingivitis when
TABLE 3: RESULT OF STABILITY STUDY OF HERBAL MOUTHWASH
Observation(weeks)
Temperature Evaluationparameters
1 2 3 4
Lightbrow Lightbrow
VisualAppearance Lightbrown Lightbrown
n n
3-5°C
PhaseSeparation Nil Nil Nil Nil
Homogeneity Good Good Good Good
Lightbrow Lightbrow
n n
Room
Temperature(25°
CRH=60%) PhaseSeparation Nil Nil Nil Nil
Lightbrow Lightbrow
n n
40°C±2°C
RH=75% PhaseSeparation Nil Nil Nil Nil
TABLE 4: RESULT OF AGAR WELL DIFFUSION ANTIBACTERIAL STUDY
Zoneofinhibition(mm)
Organism
20 μl 40 μl 60 μl 80 μl
S.aureus 7 12 15 18
E.coli 12 17 19 21
Standard Zoneofinhibition(mm)
Penicillin G 20 μl 40 μl 60 μl 80 μl
S.aureus 9 15 18 20
E.coli 13 18 20 22
Fig. 1 Antimicrobial activity of standard Penicillin G against S. aureus
Fig. 2 Antimicrobial activity of standard Penicillin G against S. E. coli
Fig. 3Antimicrobial activity of prepared mouthwash against S. aureus
Fig. 4 Antimicrobial activity of prepared mouthwash against E.coli
IV. CONCLUSION: [3]. Pathan M, Bhat K, Joshi V. Comparative

A study effort has been made to combine evaluation of the efficacy of a herbal
the active constituents of different extracts to make mouthwash and chlorhexidine mouthwash
an effective polyherbal mouthwash formulation on select periodontal pathogens: An in vitro
which is free from alcohol. For the present study and ex vitro study. J ind S per. 2017: 270-
neem, tulsi, Liquorice, cinnamon, mint turmeric 275.
and clove extracts have been used for their reported [4]. C. K. Kokate, Pharmacognosy, vol. 12,
activities including anti-bacterial activity.The NiraliPrakashan, 1999.
results of zone of inhibition also confirmed that this [5]. J. G. Hardman, L. E. Limbird, L. S.
herbal mouth rinses was found to be a potent Goodman, and A. G. Gilman, Goodman and
plaque inhibitor, and were preferred by the patients Gilman's the Pharmacological Basis of
for its taste, convenience of use and test duration in Therapeutics, McGraw Hill, New York, NY,
their mouth after rinsing. Thus, these can be used USA, 10th edition, 2001.
as an adjunct to mechanical therapy for treating [6]. Baron, J. E. and $. M. Finegold, Methods for
plaque induced gingivitis. Present study has an Testing Antimicrobial Effectiveness. In:
important impact in order to create an effective and Barley Scotts: Diagnostic Microbiology
inexpensive herbal oral health intervention for low Mosby, C.V. (Ed.). Missouri, USA, 1990;
social economic communities. However this study 171-174.
was short-term study so long term studies are [7]. Kumar P. Ansari S.H.Ali J. Herbal remedies
required with larger. The natural herbs used in for the treatment of pridental disease: A
present formulation have been medicinally proven patient review Recent pat drug deliver
to prevent the problem of oral hygiene and bad formula 2009 3:221-8.
breath. Since years and decades, these herbs have [8]. Dhar R. Zhang K, Talwar GP. Garg S,
been known for working wonders as reflected in Kumar NJ.Inhibition of the growth and
many research findings. Person can easily rinse his development of asexual and sexual stages of
mouth using this herbal mouthwash and stay clear drug-sensitive and resistant strains of the
of wide variety of oral health issues. human malaria parasite Plasmodium
falciparum by Neem (Azadirachtaindica)
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International Journal of Pharmaceutical Research and Applications

Volume 7, Issue 3 May-June 2022, pp: 720-728 www.ijprajournal.com ISSN: 2456-4494

Preparation and Evaluation of Herbal Mouthwash

TABLE 1:COMPOSITION OF HERBAL MOUTHWASH

1 Turmeric Curcuma longa Antibacterial 1ml

3 Tulsi Ocimumtenuiflorum Dental care 4ml

5 Clove Syzygiumaromaticum Anti-inflammatory 1.5 ml

6 Cinnamon Cinnamomumverum Sweetening agent 2.5ml

7 Liquorice Glycyrrhizaglabara Anti-bacterial 2 ml

Sr No Name of herbs Tests Observation

1 Neem Aq. Extract + ferric chloride 5% Dark colourisation

2 Tulsi 5 mg extract of Ocimum sanctum in Red colour

7 Mint 5 mg extract of Ocimum sanctum in Red colour

TABLE 3: RESULT OF STABILITY STUDY OF HERBAL MOUTHWASH

PhaseSeparation Nil Nil Nil Nil

Homogeneity Good Good Good Good

Homogeneity Good Good Good Good

Homogeneity Good Good Good Good

TABLE 4: RESULT OF AGAR WELL DIFFUSION ANTIBACTERIAL STUDY

Fig. 1 Antimicrobial activity of standard Penicillin G against S. aureus

Fig. 2 Antimicrobial activity of standard Penicillin G against S. E. coli

Fig. 3Antimicrobial activity of prepared mouthwash against S. aureus

Fig. 4 Antimicrobial activity of prepared mouthwash against E.coli

IV. CONCLUSION: [3]. Pathan M, Bhat K, Joshi V. Comparative

matrica, persica and chlorhexidine. J Dent [19]. Vinoth B and Manivasagaperumal R:

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