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 inhibation 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 anibiotic ciprofloxacin,
Penicillin, Tetracycline, streptomycin, Erthromycin for antibacterial and
Amphotericin B, Fluconazole, Itraconazol for antifungal pathogens.
Key words: Piper
betel leaf, Antibacterial properties, Pathogens, Methanolic extract, agar well diffusion,
also called as antibacterials, 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 is 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 is 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. It is also known as Nagaballi,
Nagurvel, Saptaseera, Sompatra, Tamalapaku, Tambul, Tambuli, Vaksha Patra, Vettilai,
Voojangalata etc in different countries (7,8). The Piper betal leaf is an
evergreen and creeper, with glossy heart heart shaped and white catkin. It is
largely distributed in tropical and subtropical region of the world. Piper
betal leaf is cultivalted in India,
Srilanka, Malaysia, Indonesia, Phillipine, Island and East Africa.The parts of
Piper betal are leaves, roots, stem, stalks and fruits. Piper betel has light
yellow aromatic essential oil, with sharp burning taste (9). It is used as a
mouth freshener after lunch and dinner in Indian families. Due to strong pungent aromatic flavour betel
leaves are used as masticatory by the Asian people. Chewing of betal leaf
produce a sense of well-being, increased alertness, sweating, salivation, hot
sensation and energetic feeling with exhilaration. It increase the capacity to
exercise physical and mental functions more efficiently for a longer duration
but it may produce a kind of psychoactive effect causing a condition of mild
addiction leading to habituation and withdrawal symptoms (8).
Sirih, sirih melayu, sirih cina,
sirihhudang, sirih carang, sirih kerakap
Betel, betel pepper, betel-vine
Sirih, surah, bodeh
composition of fresh betel leaf:
Contents of Betel
Betel leaf contain
good minerals and vitamins and also contain huge number of bioactive molecules.
Betel leaf have oil , sugar, vitamin c, and
starch. Betel leaf have phenol that is
called as chavicol that have properties
of reducing central nervous system (7,9).
Traditional uses of
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).
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).
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
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).
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).
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.
Piper betal leaf (Sanchi pan)
Plates, Flask, Tubes, Pipette, Bunsen burner, Match box, Wire loop,
weight machine, oven, nutrient agar, sabouraud dextrose agar.
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.
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 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 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.
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.
0.3% beef extract/yeast extract
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.
pH adjust to 5.6 at 250 C
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.
Dried leaves in
hot air oven for 5 minutes.
Leave flame open
for 5 minute to make your environment clear.
Take a leaves and
washed with distilled water.
Take 2gm of powdered
Now grinder the
dried leaves and makes a powder.
Soaked 2gm of
dried powder sample in 80ml methanol.
Kept in dark
place for 4 days until the secondary metabolites get dissolved.
Fourth day session:
After 4 days
filter the filtrate in petriplates by the help of What’s man filter paper
Now the dried
metabolite extract was dissolved in DMSO (Dimethyl Sulfoxide).
Take 3 nutrient
agar and 1 Sabouraud dextrose agar plates and label with each organism.
petriplates in hot air oven at 50 degree so that methanol gets evaporated.
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.
incubated at 37 degree foe 24 hours.
ON next day note
Now these wells
were filled with 60 microliter of Methanolic extract and Streptomycin
antibiotic for antibiogram.
Fifth day session:
On fifth day we perform Antibiogram analysis of tested organisms:
First clean your
table top and surrounding with alcohol dipped cotton.
Leave flame open
for 5 minute to make your environment clean.
Now place the
antibiotics on each plate in order to check the effectiveness.
On next day note
Place the plates
in incubator at 37 degree for 24 hours.
Take 3 nutrient
agar plates and label the each organism.
Antibacterial susceptibility assay of Methanolic extract of piper betel
leaf against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli
and Candida albican.
ZONE OF INHIBATION BY STREPTOMYCIN (in mm)
ZONE OF INHIBATION BY SAMPLE (in mm)