Preparation of Antibacterial Herbal Mouthwash against Oral Pathogens

17 Pages·2016·0.47 MB·English

Int.J.Curr.Microbiol.App.Sci (2016) 5(11): 205-221
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 5 Number 11 (2016) pp. 205-221

Journal homepage: http://www.ijcmas.com

Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.511.023

Preparation of Antibacterial Herbal Mouthwash against Oral Pathogens

J. Nasreen Banu* and V. Gayathri

Department of Microbiology, Ethiraj College for Women, Chennai- 600008, Tamil Nadu, India
*Corresponding author

A B S T R A C T

Medicinal plants, plays vital role in curing diseases due to their antimicrobial and

antifungal activ ity against human pathogens through decades. Herbal Mouthwashes are
in high demand , because thy act on oral pathogens and relieve the pain instantly and are
also less side e ffective. One of the most common infectious diseases encountered by
many individua ls are Dental carries and Periodontal diseases at different stages of their

life time. Dental caries include the cavity formation, eruption of enamel, swollen gums,

bleeding gums, formation of hollow black eruption on the surface of the teeth. In early

Keywords
days, Dental c aries are high among Children and Adolescents, because they do not

Antibact erial, practice proper oral hygiene. To prepare Anti-bacterial Herbal Mouthwash from the
Herbal M outhwash, aqueous extrac ts of 4 different leaves namely Azadirachta indica (Neem), Ocimum
Staphylo coccus basilicum (Tuls i), Mentha longifolia (Mint), Punica granetum Linn (Pomegranate) and
aureus, Lactobacillus rhizomes of C urcuma longa (Turmeric) that acts against the oral pathogens-
bulgaric us,
Staphylococcus aureus, Streptococcus pyogenes, Lactobacillus bulgaricus, Bacillus
Bacillus subtilis,
Streptoc occus subtilis and Esc herchia coli and to check the Anti-microbial activity by using Agar well
pyogene s, diffusion metho d. Punica granetum Linn(Pomegranate) shows sensitivity towards the
Escherc hia coli. Streptococcus pyogenes, Lactobacillus bulgaricus, Staphylococcus aureus, Bacillus
subtilis and Es cherchia coli. Azadirachta indica (Neem) shows sensitivity towards
A rticle Info
Staphylococcus aureus and Escherchia coli and shows resistance towards Streptococcus

pyogenes, Lact obacillus bulgaricus and Bacillus subtilis. Ocimum baslicum (Tulsi)
Accepte d: shows sensitivity towards Staphylococcus aureus, Bacillus subtilis and Escherchia coli
12 October 2016
and shows resistance towards Streptococcus pyogenes, Lactobacillus bulgaricus.
Available Online:
Mentha longi folia (Mint) shows sensitivity towards Staphylococcus aureus,
10 November 2016
Lactobacillus bulgaricus and Escherchia coli and shows resistance towards

Streptococcus pyogenes and Bacillus subtilis. Curcuma longa (Turmeric) shows

sensitivity towards Lactobacillus bulgaricus, Staphylococcus aureus and Escherchia coli

and shows res istance towards Streptococcus pyogenes, Bacillus subtilis. Hence this
study proves that Punica granetum Linn(Pomegranate) shows effectiveness against all
the 5 oral bacterias namely Strep tococcus pyogenes, Lactobacillus bulgaricus,
Staphylococcus aureus, Bacillus sub tilis and Escherchia coli. Pomegranate leaves
possess flavonoids which is used a s an anti-oxidant in conditions of Xerotomia.
Thereby, Pomegranate leaves can also be included in the preparation of Herbal

mouthwashes.

Introduction

The importance of herbs are highly plays vital role in curing diseases due to
considered as effective in contrast to their antimicrobial and antifungal activity
chemical products. Medicinal plants, against human pathogens through decades.
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Int.J.Curr.Microbiol.App.Sci (2016) 5(11): 205-221
Herbal Mouthwashes are in high demand, mouth rinses are used to remove the
because thy act on oral pathogens and retained food particles in a short period of
relieve the pain instantly and are also less time.
side-effective. Chemical mouthwashes have
hydrogen peroxide and chlorhexidine as an The mouth washes are concentrated
immediate whitener, steriliser and pain aqueous anti-bacterial solution that are used
reliever of teeth, but they tend to produce against oral microbes to counter oral
discoloration of teeth and may produce side infection, cleansing, to get rid of bad breath
effect, meanwhile they are cost effective. refreshing ,anti-septic .The mouthwash
One of the most common infectious plats an prominent role in the oral hygiene
diseases encountered by many individuals of an individual ,it helps to relieve
are Dental carries and Periodontal diseases symptoms of inflamed gums gingivitis. And
at different stages of their life time. also it reliably used to destruct the
pathogenic germs. The mouth washes are
Periodontal diseases can lead to destruction used by most of the dental patients to
of ligament, cementum, gingiva and overcome sour mouth (xerostomia),
alveolar bone. Plaque is the main ulcerated throat and sensitive teeth. Dentists
etiological causes for the gingival always use mouthwash as an antimicrobial
inflammation. Thus the control of the agent before oral surgery of the patients,
plaque can be done by using instant herbal because they help to sterilise the surface of
mouth wash. Mouth washes have the ability the inflamed gums and teeth, thereby the
to deliver the therapatic ingredients and contamination of any other microorganisms
ingredients to access against the organism can be avoided.
present on the surface of the mouth.
Cholorhexidine is regarded as gold standard The Pomegranate (Punica granatum L.) is
mouth wash but has a significant side an ancient fruit. The pomegranate belongs
effects, apart from staining the teeth after to the family Punicaceae. It is native from
long term use, like contact dermatitis, IgE the area of Iran to the Himalayas in
mediated hypersensitivity (Monica Lamba, northern India, and has been cultivated and
2015). naturalized over the entire Mediterranean
region since ancient times. A clinical
The role of junk foods in affecting the oral analyses determine the leaf mineral
cavity of an individual is high and contents (N, P, K, Ca, Mg and Fe) were
unavoidable. The foods like Candies, significantly affected the organisms with
chocolates, jellies and jams have high sugar anti-oxidant treatments. The pomegranate
content the children and adolescents are extracts shows effective reduction of
usually prone to consume this kind of sugar pathogens in chronic periodontal disorders.
products but ,the sugar content possess The pomegranate are anti-inflamatory in
insoluble glucan which gets attached to the nature, hence they are used to treat against
enamel of the tooth resulting in the oral-ailments. The pomegranate possess
formation of cavity in tooth. The active polyphenolic flavonoids like
carbonated drinks is other important punicalagins and ellagic acids which helps
destroyer of teeth enamel, as it erodes the to prevent gingivitis and or-potential of the
enamel some may even results in depth mouth.
eruption of dentine and results in tooth
The Mentha leaves (Mentha longifolia) are
discolouration. Hence mouthwashes or
aromatic, perennial herbs. The mentha
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Int.J.Curr.Microbiol.App.Sci (2016) 5(11): 205-221
belongs to the family Lamiaceae. 0.1% mercuric chloride, thus it helps in the
Peppermint was first described in 1753 by instant whitening of teeth. The basil leaves
Carl Linnaeus. Mentha are extensively used shows high microbial activity against
as flavouring ingredient in breath freshness, Streptococcus pyogenes, Staphylococcus
antiseptic mouth rinses, chewing gums and aureus and E.coli.
tooth paste. The pulegone primarily
responsible for the aroma and flavour of Turmeric (Curcuma longa) a rhizomatous,
spearmint is L-Carvone. Menthol oil herbaceous plant of the ginger family
contains menthone, menthyl esters, menthyl zingiberaceae. It is native to south west
acetate and menthofuran. Mint also consists India requiring temperatures between 20
of small amounts of many additional and 30 C and the plants are gathered
compounds including Limonene, Pulegone, anually for their rhizomes. Turmeric was
Caryophyllene and Pinene. first used as a dye by ancients, then later for
its medicinal properties. The important
The use of Neem are known from ancient chemical components of turmeric are a
dates back since 45,000 years against oral group of compounds called curcuminoids
diseases. The neem leaves Azadirachta that includes demethoxycurcumin and
indica belongs to the family Lamiaceae. bisdemethoxycurcumin. The other volatile
The neem solutions are used in decreasing compounds include turmerone, altantone
the inflammation of gums, to remove and zingiberine. According to the National
canker and against dental cavities. centre for complementary and Integrative
Nowadays the neem extracts are used as health, some researches shows compound in
antiseptic substance against inflammation turmeric to have antifungal and
of mouth. The neem extract shows antibacterial properties.
significant effects on both Gram positive
and Gram negative bacteria such as Chlorhexidine is a chemical antiseptic
Staphylococcus spp, Streptococcus spp, solution against Gram positive and negative
E.coli and Salmonella. bacteria, aerobic and anaerobic spp. It is
used in dental surgery to create germ-free
A.indicahas inhibition against the environment, periodontal treatment,
production of insoluble glucan,which is the halitosis and Xerostomia. It is highly used
main cause for plaque formation.Theneem in the dental disorders patients especially,
sticks has proven to show markedly individuals using permanent braces.
inhibition against Streptococci ,which Turmeric oil plays a vital role in killing oral
colonise the surface of the tooth. cancer cells. Curcumin loaded nano-
particles were used in chemotherapy
The Basil leaves (Ocimumbasilicum L.) is resistant against oral submucous fibrosis.
an annual herbs, which is used as an Turmeric extracts can replace
antimicrobial and antifungal substance chlorhexidine, as it helps to reduce burning
against oral microbes. The basil leaves are sensation, staining of teeth, allergy-type
used by Hindus since, dates back for symptoms, numbness of teeth and destruct
reducing morning bad breathe. It consists of plaque forming bacteria.
phytochemical compound Terpenes, cineole
and other allophatic compounds. It also The aim to prepare Anti-bacterial Herbal
consists of Linalool and methyl eugenol as Mouthwash from the aqueous extracts of 4
essential oil. The basil leaves naturally has different leaves namely Azadirachta
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indica(Neem), Ocimum basilicum(Tulsi), Collection of organisms
Mentha longifolia(Mint), Punica granetum
Linn (Pomegranate) and rhizomes of Oral swab: The sterile absorbent cotton
Curcuma longa (Turmeric) that acts against was rolled against the tip of the wooden
the oral pathogens- Staphylococcus aureus, stick besides the flame to avoid spores and
Streptococcus pyogenes, Lactobacillus were sterilised by autoclaving at 121ºC for
bulgaricus, Bacillus subtilis and Escherchia 15 mins. The swab was rubbed on to the
coli and to check the Anti-microbial activity gums and periodontal region of both upper
by using Agar well diffusion method. and lower jaws of the individuals. Totally
25 oral swabs were collected from the
The present study objectives includes that individuals under sterile condition inside
to prepare the aqueous extracts of the laboratory.
Azadirachta indica (Neem), Ocimum
basilicum Tulsi), Mentha longifolia(Mint), Peptone water:1.5g of peptone water was
Punica granetum Linn (Pomegranate) and weighed and dissolved in 100ml of distilled
Curcuma longa ( Turmeric ). water, mixed gently and were autoclaved at
121ºC for 15mins.Cooled to 50-55ºC and
To isolate and study the colony morphology
transferred to sterile test tubes, the oral
of the oral microorganisms-Staphylococcus
swabs were inoculated and incubated at
aureus, Streptococcus pyogenes,
37ºC for 1h and results were observed.
Lactobacillus bulgaricus, Bacillus subtilis

and Escherichia coli by using culture media
Maintaining the culture: The culture was
and biochemical tests.
allowed to store in Refrigerator and

frequently subcultured by inoculating it in
To perform the agar well diffusion
Nutrient slant or Peptone water for long
technique in the Muller Hinton agar and to
period of usage.
identify the anti-bacterial activity of leaf

extracts against test organisms,by
Collection of Plant Leaves: Leaves of
measuring the zone of inhibition.
Azadirachtaindica(Neem),

Ocimumteniflorum(Tulsi), Mentha
To compare the efficacy of extracts of 4
longifolia(Mint), Punicagranetum
different herbal leaves on the basis of their
Linn(Pomegranate) and rhizomes of
resistancy and sensitivity towards the oral
Curcuma longa (Turmeric) were randomly
microbes and to formulate the herbal
collected from mature plants.
mouthwash.

Extraction process: The leaves were
Materials and Methods
washed with sterile water,shadow-

dried,pulverized and stored in air-tight
Test organisms: The isolates of Mouth
bottles. The Aqueous extracts were
organisms like Staphylococcus aureus,
prepared by soaking the powdered leaves in
Streptococcus pyogenes, Lactobacillus
sterile distilled water and maintained in
bulgaricus, Bacillus subtilis and Escherchia
Incubator at 37°C for 72 h and were filtered
coli, were collected by using sterile oral
using Whattmann filter paper and used for
swab under aseptical condition. They were
identifying organisms
sub-cultured in peptone water and

maintained in refrigerator for longer period
Equipments: Sterile Petriplates, Testtubes,
of storage.
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Conicalflask, Whattmann filterpaper, (iv) After 5 days, the dried leaves were
Incubator, Autoclave, Laminar air flow, taken and powdered by using sterile mixer
Pippetting device, Hotair-oven. under aseptic condition.

Preliminary test for organisms: The (v) The pulverized leaves are
collected samples were cultivated and the transferred to air-tight sterile container jars.
colony morphology on the culture media -
Nutrient agar were examined. A colony was (vi) 100ml of sterile distilled water was
picked, inoculated in peptone water and taken in 4 conical flask (250 ml), the
incubated at 37°C for 1h.The isolates were pulverised leaves were weighed and
streaked on specific medium- Blood agar, suspended in distilled water under sterile
Macconkey agar, Manitol salt agar, Eosine condition.
methylene blue and Lactobacillus Mann
rogosa agar. Meanwhile, the isolates were (vii) The preparation was heat sterilised
identified with Gram staining techniques, at 40°C for 5-10 mins and was kept for
Motility test and biochemical tests. incubation at 37°C for 72h.

Antibacterial activity of Herbal leaves (viii) After incubation, the extracts were
filtered with the help of a sterile
The antibacterial activity of Aqueous Whattmann filter paper no: 1 and a funnel
extracts of Azadirachtaindica, under lab condition.
Ocimumbasilicum, Mentha longifolia,
Punicagranetum Linn and rhizomes of (ix)The filtered extracts are boiled
Curcuma longa was determined against the vigorously again to kill the bacterial spores,
test organism-Lactobacillus bulgaricus, which will prevent from contamination.
Staphylococcus aureus, Streptococcus
pyogenes, Bacillus subtilis ,Eschrechia coli (x) The extracts after heating is ready to use
by agar well diffusion method. for the formulation of Mouth wash and also
can be tested against the oral pathogens by
Aqueous extracts of Leaves by Shadow using Agar well diffusion techniques.
drying technique
Collection of organisms
(i) The leaves of mature plants were
collected and washed 3-5 times with tap (i) 25 students were selected randomly
water to remove dust and dirt. for the collection of mouth organisms.

(ii) The leaves were allowed to soak in (ii) The oral swabs were rubbed on the
already boiled water bath at 30-40ºC for 10- periodontal region of their mouth gently,
15 mins to kill microbes present in the under aseptic condition.
surface of the leaves.
(iii) The collected swabs were inoculated
(iii) The leaves undergoes Shadow in freshly prepared saline or peptone water
drying technique were the leaves were and incubated at 37°C for 2h.
spread in sterile container trays and kept at
ambient temperature for 5 days.

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(iv) The luxuriantly grown organisms (ii) 19.0g of agar was weighed and
were isolated and identified in culture dissolved in the water and a pinch of agar-
media or by using biochemical techniques. agar was added for solidifying the media.

(v) After using the culture, it was (iii) The media was autoclaved at 121°C
refrigerated till the next use, meanwhile for 15 mins, cool to 55°C.
before using the culture, they were
incubated at 37° C for 2 h. (iv) 5-7ml of agar was poured in the 25
petriplates, to form a thick agar plate and
Isolation of Organisms kept in room temperature to get settled.

(i) 25 plates of nutrient agar was (v) The plates were then air dried and
prepared by above mentioned procedure, UV radiated in the laminar air flow for 5-10
each plates were inoculated with 25 mins.
swabbed culture by using Streak plate
technique and was incubated at 37°C for 24 (vi) The agar wells were punctured by
h. using sterile micropipette tips with equal
intervals and the dilutions were marked as
(ii) After incubation, the 2-3 different 50µl, 100µl, 150µl for each extracts.
colonies were observed in the plates, 5
randomly selected colonies were picked out (vii) The isolated bacterial colonies were
and transferred to the freshly prepared picked from the test organism culture media
saline and incubated at 37°C for 2h. and inoculated in peptone water or saline
and was incubated at 37°C for 2h.
(iii) The isolates after incubation was
identified by preliminary test such as Gram (viii)After incubation the bacterial cultures
staining technique, Motility test, Catalase were compared with 0.5 Mac Farland's
and Oxidase test. standard, for the standardization of the
bacterial cultures.
(iv)The isolates were streaked on the
specific agar plates such as Blood agar, (ix) With the help of oral swab the isolates
Macconkey agar, Emb agar and were swabbed on the upper surface of the
Lactobacillus MRS agar and also agar gently, under sterile condition.
inoculated into biochemical tubes under
sterile condition which was then, incubated (x)The micro pipette is used to transfer the
at 37°C for 24h. aqueous extracts in each wells and
incubated at 37°C for 24 h.
(v) After incubation at 37°C for 24h, the
plates were observed for colony (xi) After 24h, the plates shows agar
morphology and the isolates were diffusion around the wells and the results
differentiated by identifying the bio- were identified by measuring the zone of
chemicals. inhibition.

Agar well diffusion technique
(xii) The zone of inhibition was

identified for the resistance of the
(i) A sterile conical flask was taken and
organisms.
filled with 500 ml of distilled water.
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Results and Discussion media and biochemical techniques. The test
organisms were isolated and identified and
Aqueous extraction of herbal leaves
the results were recorded based on the

The aqueous extracts of the leaves were morphological and cultural characters.
done by suspending shadow dried and
pulverized leaf powder in sterile distilled Anti-bacterial activity
water. The leaf extracts were subjected to
heat, vigorously to kill all the aerobes and The Herbal mouthwash prepared by the
spores, there by the extracts are free from aqueous extract from the leaves of the herbs
contaminants. On the other hand, the shows:
component of leaf and its antigenicity were
The Herbal mouth wash was prepared from
not lost by heating. The aqueous extract is
4 different leaves- Neem (Azadirachta
purely herbal hence no chemical component
indica), Pomegranate (Punica granetum
is involved in the preparation of the
Linn), Basil (Ocimum baslicum) and Mint
extracts. The aqueous extracts were
(Mentha longifolia) by Aqueous extraction
prepared as it shows very less side-effects
method, thus the mouthwash was purely,
than the other commercially available
herbal and contains no chemical ingredient.
chemical mouth washes. The herbal mouth
And also the leaves were allowed to
washes are homemade hence it is of low
shadow dried hence the phytochemical
expenditure whereas, the commercially
component and the antibacterial activity of
available mouthwash is cost-effective. The
the leaves are not lost.
herbal mouthwash has short shelf life when

compared to chemical mouthwash but the
The isolates of the oral test organisms-
storage of unsealed mouthwash may tend to
Staphylococcus aureus, Streptococcus
invade the air microbes and other
pyogenes, Lactobacillus bulgaricus,
facultative anaerobes.
Bacillus subtilis and Escherchia coli were

The extracts of Neem is taken very less idententified and isolated by using Streak
amount due to its bitterness, it acts as Anti plate method in culture media like Nutrient
inflammatory component against bleeding agar, Blood agar, Macconkey agar, Eosine
gums. Mint is known for its aroma hence it methylene blue agar and Lactobacillus
helps to get rid of bad breath. Pomegranate MRS agar.
leaves possess flavonoids which is used as
an antioxidant in conditions of Xerostomia. The extracts were taken in different
Tulsi extracts were used as they consists of dilutions and inoculated against the
little amount of mercury chloride as a leaf swabbed test isolates in Mueller Hinton
component, which acts as natural whitener. agar and the sensitivity and resistance of the
It helps in destruction of oral microbes by test organism against the formulated mouth
preventing oral disorders like pyorrhea and wash were analysed. As by their
cavities. Turmeric is used to replace effectiveness the dilution of the extracts
chlorhexidine, it is an anti-microbial and were formulated.
anti-septic agent used in oral hygiene.
Punica granetum Linn (Pomegranate)
Isolation of test organisms shows sensitivity towards the Streptococcus
pyogenes, Lactobacillus bulgaricus,
The isolates from the collected oral swabs Staphylococcus aureus, Bacillus subtilis
were identified with the help of culture and Escherchia coli. Hence this study
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Int.J.Curr.Microbiol.App.Sci (2016) 5(11): 205-221
proves that Punica granetum Linn bulgaricus, Staphylococcus aureus,
(Pomegranate) shows effectiveness against Bacillus subtilis and Escherchia coli
all the 5 oral bacterias namely (Table.1-5 and Figure.1-6).
Streptococcus pyogenes, Lactobacillus

Table.1 Zone of inhibition in Neem Extracts

S.no ORAL MICROBES DILUTION ZONE OF SENSITIVITY RESISTANCE
OF THE INHIBITION
EXTRACTS
1 Staphylococcus 50 µl 7 mm + -
100 µl 10 mm + -
150 µl 15 mm + -
2 S. pyogenes 50 µl 18 mm - +
100 µl 22 mm - +
150 µl 24 mm - +
3 Escherchia coli 50 µl 17 mm + -
100 µl 21 mm + -
150 µl 25 mm + -
4 L. bulgaricus 50 µl 9 mm - +
100 µl 10 mm - +
150 µl 12 mm - +
5 Bacillus subitilis 50 µl 5 mm - +
100 µl 8 mm - +
150 µl 10 mm - +

Table.2 Zone of inhibition in Tulsi Extracts

S.no ORAL MICROBES DILUTION ZONE OF SENSITIVITY RESISTANCE
OF THE EXTRACTS INHIBITION
1 Staphylococcus aureus 50 µl 18mm + -
100 µl 20 mm + -
150 µl 23 mm + -
2 Streptococcus pyogenes 50 µl 10 mm - +
100 µl 14 mm - +
150 µl 19 mm - +
3 Escherchia coli 50 µl 15 mm + -
100 µl 19 mm + -
150 µl 25 mm + -
4 L.bulgaricus 50 µl 11 mm - +
100 µl 13 mm - +
150 µl 16 mm - +
5 Bacillus subitilis 50 µl 10 mm + -
100 µl 12 mm + -
150 µl 18 mm + -
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Table.3 Zone of inhibition in Mint Extracts

S.no ORAL MICROBES DILUTION ZONE OF SENSITIVITY RESISTANCE
OF THE INHIBITION
EXTRACTS
1 Staphylococcus aureus 50 µl 16 mm + -
100 µl 17 mm + -
150 µl 22 mm + -
2 Streptococcus pyogenes 50 µl 9 mm - +
100 µl 10 mm - +
150 µl 12 mm - +
3 Escherchia coli 50 µl 15 mm + -
100 µl 18 mm + -
150 µl 20 mm + -
4 L.bulgaricus 50 µl 9 mm + -
100 µl 11 mm + -
150 µl 17 mm + -
5 Bacillus subitilis 50 µl 9 mm - +
100 µl 10 mm - +
150 µl 12 mm - +

Table.4 Zone of inhibition in Pomegranate Extracts

S.no ORAL MICROBES DILUTION ZONE OF SENSITIVITY RESISTANCE
OF THE INHIBITION
EXTRACTS
1 Staphylococcus aureus 50 µl 23mm + -
100 µl 26mm + -
150 µl 33 mm + -
2 Streptococcus pyogenes 50 µl 21 mm + -
100 µl 23 mm + -
150 µl 25 mm + -
3 Escherchia coli 50 µl 20 mm + -
100 µl 24 mm + -
150 µl 25 mm + -
4 L.bulgaricus 50 µl 22 mm + -
100 µl 24 mm + -
150 µl 31 mm + -
5 Bacillus subitilis 50 µl 20 mm + -
100 µl 21 mm + -
150 µl 23 mm + -

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Table.5 Zone of inhibition in formulated Herbal Mouthwash

S.no ORAL MICROBES DILUTION ZONE OF SENSITIVITY RESISTANCE
OF THE INHIBITION
EXTRACTS
1 Staphylococcus aureus 50 µl 11 mm - +
100 µl 10 mm - +
150 µl 16 mm - +
2 S. pyogenes 50 µl 11 mm - +
100 µl 12 mm - +
150 µl 15 mm - +
3 Escherchia coli 50 µl 11 mm - +
100 µl 14 mm - +
150 µl 15 mm - +
4 L.bulgaricus 50 µl 9 mm - +
100 µl 15 mm - +
150 µl 16 mm - +
5 Bacillus subitilis 50 µl 8 mm + -
100 µl 11 mm + -
150 µl 13 mm + -

Fig.1 Antimicrobial activity of Pomegranate extracts.

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