Abstract:

The study of
antimicrobial properties of Piper betel leaf which are cultivated in India .The
Methanolic extract of dried leaf  were tested
for antibacterial and antifungal properties against pathogenic microorganisms
such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and
Candida albican. Methanolic extract showed effective zone of inhibition against
the used pathogens. 25mm against Staphylococcus aureus followed by the zone of
16mm against Pseudomonas aeruginosa followed by the zone of 20mm against
Escherichia coli and 17mm against Candida albican by using agar well diffusion
method. Antibiogram was also be done against these pathogenic bacteria by using
antibiotic ciprofloxacin, Penicillin, Tetracycline, streptomycin, Erythromycin
for antibacterial and Amphotericin B, Fluconazole, Itraconazol for antifungal
pathogens.

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Key words: Piper
betel leaf, Antibacterial properties, Pathogens, Methanolic extract, agar well
diffusion, Antibiogram.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introduction:

 

Antibiotics are
also called as antibacterial, and are a type of antimicrobial drug used in the
treatment and prevention of bacterial and fungal infection. They may either
kill or inhibit the growth of bacteria. Sometimes the term antibiotics are used
to refer any substance used against microbes. Antibiotics revolutionized
medicine in 20th century. Using antibiotics to eliminate the infection produce
adverse effect to host organs, tissues and cells. The effect produced by the
antimicrobial agents can be prevented or cure with herbal medicines. Herbal
medicines are safe, and will overcome the resistance produced by pathogens.
Some medicinal herbs have antibacterial and antifungal properties which will be
useful for the clinical use (1,3). Herbal medicines are a traditional method of
fighting against pathogens. The herbal medicines or medicinal plants have been used
since time immemorial in Indian villages for the treatment of uncountable
diseases (2).

 

Piper betel Leaf
belongs to family Piperaceae commonly known as Pan i.e. the Black Pepper family
it is traditionally used in India, China, Thailand (7,8). The Piper betel leaf
is green in color and has aromatic smell, it is bitter in taste and it is heart
shaped. It is largely distributed in tropical and subtropical region of the
world. For mouth freshener the betel leaf is the best freshener that can be
taken after lunch or dinner (9).  It has
great capacity to increase are physical health and mental function. In English it
is called betel, betel vine and betel piper and in Hindi it is call pan (8).

Nutritional
composition of fresh betel leaf:

                     S. No.

             Constituents

           Approximate
           composition

1

Water

85-90%

2

Protein

3-3.5%

3

Fat

0.4-1.0%

4

Mineral

2.3-3.3%

5

Fiber

2.3%

6

Chlorophyll

0.01-0.25%

7

Carbohydrate

0.5-6.10%

8

Nicotinic acid

0.63-0.89mg/100g

9

Vitamin C

0.005-0.01%

10

Vitamin A

1.9-2.9mg/100g

11

Thiamine

10-70mg/100g

12

Riboflavin

1.9-30mg/100g

13

Tannin

0.1-1.3%

14

Nitrogen

2.0-7.0%

15

Phosphorus

0.05-0.6%

16

Potassium

1.1-4.6%

17

Calcium

0.2-0.5%

18

Iron

0.005-0.007%

19

Iodine

3.4mg/100g

20

Essential Oil

0.08-0.2%

21

Energy

44 kcal/100g

 

Contents of Betel
leaves:

Betel leaves
contain good minerals and vitamins and also contain huge number of bioactive
molecules. Betel leaf has oil, sugar, vitamin c, and starch.  Betel leaf has phenol that is called as
chavicol that have properties of reducing central nervous system (7, 9).

Traditional uses of
Betel leaves:

Betel leaf is
traditionally known to be useful for the treatment of various diseases like bad
breath, boils and abscesses, conjunctivitis, constipation, headache, hysteria,
itches, mastitis, mastoiditis, leucorrhoea, otorrhoea, ringworm, swelling of
gum, rheumatism, abrasion, cuts and injuries , wound and inflamation etc as
folk medicine while the root is known for its female contraceptive effects
(2,6).

Anti-inflammatory
effects:

Piper betal leaf
possess anti-inflamatory effects in various animal models of studies with
various inflamogens. Betal leaf used as a common home remedy for infalmmation
in the oral cavity (7).

Antioxidant
effects:

The contents of
Piper betal leaf extract increased the cellular antioxidants and mediate the
chemopreventive effects at least in part. Three varities of Piper betal leaf
showed   antioxidant effects when
evaluated by in vitro systems such as DPPH radical scavenging, superoxide
radical scavenging, hydroxyl radical scavenging and prevention of lipid
peroxidation (7,9).

Antimicrobial
Activity:

The antimicrobial
activity of leaves towards bacteria in mouth 
i.e. Streptococcus viridans, Staphylococcus aureus and Streptococcus
mutans and heals many bacterial diseases and also show antimicrobial activity
against various obligate oral anaerobes. The methanolic extract was more
effective than other extracts in inhibating the microbial organisms. Piper
betal leaf is most active antimicrobial plant (3,7,9).

 

Antifungal
activity:

The extract of
Piper betel L., (Piperaceae) is used from which the Hydroxychavicol, isolated
from the chloroform extraction to isolate the antifungal activity agaist the
selected fungi. Hydroxychavicol compound can be used as an antifungal agents to
exhibated the antifugal activity and also used to treat topical infections as
well as gargle mouthwash against oral Candida infections (7).

 

Anti-diabetic
activities:

Methanolic extract
of Piper betal leaf possess 
hypoglycaemic activity when tested in normoglycaemic rats using hot
water and cold water extracts (9).

 

Role of betel leaf
extract on thyroid function:

The effect of betal
leaf extract depend on the thyroid hormones concentrations lipid peroxidation
(LPO) and on the activities of superoxide dismutase (SOD) and catalase (CAT).
Administration of betel leaf extract exhibited a dual role, dependingon the
different doses. While the lowest dose decreased thyroxine (T4) and increased
serum triiodothyronine (T3) concentrations, reverse effects were observed at
two higher doses. Lipid peroxidation (LPO) increased with higher doses and
decreased in superoxide dismutase (SOD) and catalase (CAT) activities. However
, most of these effects were reversed with lowest dose. Betel leaf can be both
stimulatory and inhibitory to thyroid function, particularly for T3 generation
and lipid peroxidation in male mice, depending on the amount consumed (7).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Materials and Methods.

Sample:

Piper betal leaf (Sanchi pan)

Apparatus requirement:

Plates, Flask, Tubes, Pipette, Bunsen burner, Match box, Wire loop,
weight machine, oven, nutrient agar, sabouraud dextrose agar.

Orgainsms:

·        
Pseudomonas
aeruginosa

·        
Staphylooccus
aureus

·        
Escherichia coli

·        
Candida albicans.

Pseudomonas aeruginosa:

P.aeruginosa is a gram negative organism an a rod shaped bacterium. It
cause diseases in animal, plants as well as humans. It occur especially in
patients with compromised immune system. It is commonly found in soil, water
and moist enviroment.

Staphylococcus aureus:

S.aureus is a Gram positive bacteria 
and it is a round shaped bacterium. It is ommnonly found in skin, nose
and respiratory track. It is the leading cause of skin and soft tissue
infections such as abscesses, furuncles and cellulitis.The infection of S.aureus
are not serious but it can serious infection such as pneumonia, blood stream
infection, bone and joint infections.

Escherichia coli:

Escherichia coli is agram nagative organism, it is a rod shaped
bacteria. Ecoli found in food, environment and intestine of humans and animals.
It is harmless and beneficial floral of gut. By eating contaminated food or
drinking water so some strains of Ecoli can cause diarrhea.

Candida albicans:

Candida albicans is an opportunistic yeast  and it is a member of human gut flora, it
does’nt exist outside the body. It is commonly found in gastrointestinal track
and in mucous membranes such as the vagina, mouth of healthy adults, or rectum.
But it can be pathogenic in immunocompromised patients.

 

 

 

 

Agars:

Nutrient agar

Sabouraud dextrose agar

Nutrient agar:

It is a basic growth medium used for the routine cultivation of non
fastidious organism. It is useful because it doesn’t lose consistency and
remain in solid state evev at high temperature. We can easily observe bacterial
growth because of its clear surface. Different types of organism can easily
grow on nutrient agar but some bacteria cannot grow on this.

Composition:

0.5% peptone

0.3% beef extract/yeast extract

1.5% agar

0.5% NaCl

Distilled water

pH 6.8 at 25C

Sabouraud dextrose agar:

SDA media is used to cultivating pathogenic fungi, and also
determining the microbial and fungal content of cosmetics. Dextrose is the
fermentable carbohydrate incorporated in high amount of energy and carbon
source. Peptone mixture give vitamins, minerals, nitrogen and amino acid
essential for growth. The concentration 
of dextrose is high and acidic pH make this medium suitable for fungi.

Composition:

10gm peptone

40gm Dextrose

15gm Agar

Distilled water

pH adjust to 5.6 at 250 C

 

 

 

 

 

Protocol:

First day session:

First clean your
table top and surrounding with alcohol dipped cotton.

First clean your table top and
surrounding with alcohol dipped cotton.

 

Leave flame open
for 5 minute to make your environment clear.

 

 

 

 

 

 

 

Dried leaves in
hot air oven for 5 minutes.

Take a leaves and
washed with distilled water.

 

 

 

 

 

 

                                                         

Take 2gm of
powdered dried sample.

Soaked 2gm of
dried powder sample in 80ml methanol.

Kept in dark
place for 4 days until the secondary metabolites get dissolved.

Take 80ml
methanol.

Now grinder the
dried leaves and makes a powder.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fourth day session:

After 4 days
filter the filtrate in petriplates by the help of What’s man filter paper
No.1.

Now the dried
metabolite extract was dissolved in DMSO (Dimethyl Sulfoxide).

Nutrient agar and
SDA agar plates were spreaded with 60 microliter of bacterial culture to
check the antimicrobial activity.

Make 2 wells of
5mm diameter on each plate with the help of sterile borer.

Plates were
incubated at 37 degree foe 24 hours.

ON next day note
the results.

Now these wells
were filled with 60 microliter of Methanolic extract and Streptomycin
antibiotic for antibiogram.

Take 3 nutrient
agar and 1 Sabouraud dextrose agar plates and label with each organism.

Kept the
petriplates in hot air oven at 50 degree so that methanol gets evaporated.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fifth day session:

On fifth day we perform Antibiogram analysis of tested organisms:

First clean your
table top and surrounding with alcohol dipped cotton.

Take 3 nutrient
agar plates and label the each organism.

Place the plates
in incubator at 37 degree for 24 hours.

Now place the
antibiotics on each plate in order to check the effectiveness.

Leave flame open
for 5 minute to make your environment clean.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

On next day note
the results.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Observation:

Antibacterial susceptibility assay of Methanolic extract of piper betel
leaf against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli
and Candida albican.

TEST ORGANISM

ZONE OF INHIBATION BY STREPTOMYCIN (in mm)

ZONE OF INHIBATION BY SAMPLE (in mm)

Pseudomonas aeruginosa

29mm

16mm

Staphylococcus aureus

35mm

25mm

Escherichia coli

30mm

20mm

Candida albicans

______  

17mm

 

 

 

 

 

     

 

 

 

 

 

 

 

 

 

 

 

             

                           
Candida albican                                                                                    
Escherichia coli

 

 

 

                      

           
Pseudomonas aeruginosa                                                                       
Staphylococcus aureus

 

 

 

Antibiogram analysis:

Antibiotics

Pseudomonas aeruginosa

Staphylococcus aureus

Escherichia coli

Streptomycin

18mm

14mm

16mm

Tetracycline

4mm

5mm

____

Penicillin

No zone

No zone

No zone

Erythromycin

No zone

10mm

25mm

Ciprofloxacin

30mm

29mm

27mm

 

              

               

 

 

 

 

Results:

1 sample of Piper betel leaf has been taken from the market.
The sample was preceded for the analysis of antibacterial and antifungal
activity of extract of Piper betel leaf. The sample first dried and then make
powder of dried leaves of 2gm were soaked in 80ml methanol and incubated for 4
days. After 4 days filter the extract in plate and dried the extract in hot air
oven so methanol get evaporated. Now dried metabolites dissolved in DMSO to
make the extract. Now the organism was cultured on nutrient agar and on
sabouraud dextrose agar. Make wells by the help of borer and filled the wells
with extract after one day incubation the zone of inhibition observed against Pseudomonas
aeruginosa, Staphylococcus aureus, Escherichia coli and candida albican.
Pseudomonas aeruginosa give 16mm diameter of zone, Staphylococcus aureus give
25mm diameter of zone, Escherichia coli give 20mm diameter of zone and Candida
albican give 17mm diameter of zone. The result of Antibiogram against the
tested organism showed that Pseudomonas aeruginosa inhibit Ciprofloxacin and
give 30mm zone by streptomycin 18mm and by tetracycline 4mm zone.
Staphylococcus aureus inhibit Ciprofloxacin and give 29mm zone, by Streptomycin
14mm zone, Erythromycin 10mm zone and by Tetracycline 5mm zone. Escherichia
coli inhibit Ciprofloxacin and give 16mm zone, by Streptomycin 16mm zone and by
erythromycin give 25mm zone.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Discussion:

 

Piper betel leaf is a herbal medicine and it is medically
valuable. It is the best alternative antibiotic that is available against the
organisms. It is in green color with aromatic smell and bitter in taste. For
mouth freshener the betel leaf is the best freshener that can be taken after
lunch or dinner.

Methanolic extract of Piper betel leaves were prepared that
can be used for antimicrobial studies in the research work.

Agar well diffusion method of Kirby borer is used to observe
the antibacterial and antifungal properties of Piper betel leaf extract against
the pathogenic organisms.

The methanolic extract of Piper betel leaf has good
antibacterial properties against Staphylococcus aureus that showed zone of
inhibition (25mm) and Escherichia coli showed (20mm) zone of inhibition.

Ciprofloxacin antibiotic that give large zone of inhibition
against Pseudomonas aeruginosa (30mm), against Staphylococcus aureus (29mm) and
Escherichia coli (27mm) zone.

Streptomycin is the second antibiotic that can give large
zone and inhibit by these organism against Pseudomonas (18mm), against
Staphylococcus aureus (14mm) and against Escherichia coli (16mm).

Piper betel leaf can be a good source of herbal medicine
especially in methanol as a solvent. The methanolic extract can be effective
against the above bacteria.

 

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