Abstract: pathogens. Key words: Piper betel leaf, Antibacterial properties,

Abstract:

The study of
antimicrobial properties of Piper betel leaf which are cultivated in India .The
Methanolic extract of piper betel leaf were screened for antibacterial and
antifungal properties aganist 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.

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introduction:

 

Antibiotics are
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).

 

 

 

 

 

 

 

 

 

 

 

Vernacular Names:
 

 

Malaysia

 Sirih, sirih melayu, sirih cina,
sirihhudang, sirih carang, sirih kerakap

English

Betel, betel pepper, betel-vine

Tamil

Vetrilai

Hindi

Pan

Sakai

 Jera

Thai

Pelu

Javanese

Sirih, surah, bodeh

 

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

 

 

 

 

 

 

Chemical
Constituents:

Piper betal leaf
contain large number of bioactive molecule like polyphenol, alkaloids,
steroids, saponin and tannin. Leaves contains essential amino acids except
lycine, histidine and arginine. Leaf also contain minerals and good vitamins
like nicotinic acid, ascorbic acid and carotin. (7,9).

Contents of Betel
leaves:

Piper betal leaves
contain essential oil tannin, sugar, vitamin c, starch and diastase. It
contains a phenol called chavicol which possess the properties of reducing the
central nervous stimulation, sialogogue and local anaesthesia and also has a
powerfull antiseptic properties (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:

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.

 

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.

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                         

Now grinder the
dried leaves and makes a powder.

 

 

 

 

 

 

 

 

 

 

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.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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).

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

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

ON next day note
the results.

Plates were
incubated at 37 degree foe 24 hours.

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.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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.

On next day note
the results.

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

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

 

              

               

 

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