Antibiotic

resistance
 Common
Bacterial Skin
Infections
 Impetigo
Cellulitis
 Erysipelas

Folliculitis
Furuncle & Carbuncle
 FORMULATING
CONTROLLING DEVELOPING MODERN
MEDICATION
 ANTIOXIDANT ANTIMALARIAL

CYTOTOXIC ANTIFUNGAL

Piper betle Linn. ANTISEPTIC ANTIBACTERIAL

(Ikmo)
 Main Objective
The main thrust of the study is to
formulate a topical antibacterial
ointment using Ethanolic crude
extract from Piper betle Linn.
(Ikmo) Leaves.
Specific Objectives
1. To obtain ethanolic extract from Ikmo leaves and determine the
percentage yield;

2. To evaluate the ethanolic leaf extract using organoleptic,

solubility and chemical test (determination of metabolites);

3. To determine the concentration of ethanolic crude extract that

exerted significant antibacterial activity in Staphylococcus aureus
and Pseudomonas aeruginosa in comparison with the reference
standard, Mupirocin ointment.

4. To prepare and evaluate topical antibacterial ointment using

Antibacterial Susceptibility Test and Dermal IrritationTest.

5. To compare the 60% ethanolic crude extract of Ikmo leaves

against Ikmo ointment.
To determine the percentage yield of the ethanolic crude extract from
the leaves of the Piper betle Linn.
To determine the percentage yield of the ethanolic crude extract from
the leaves of the Piper betle Linn.

Weight of Crude extract

Percent yield = Weight of dried Piper betle leaves x 100
To evaluate the ethanolic crude extract from the leaves of the Piper
betle Linn.

Appearance
Color
Odor
To evaluate the ethanolic crude extract from the leaves of the Piper
betle Linn.

I. Carbohydrates III. Flavonoids VI. Alkaloids

• Molisch’s Test • Wilstater “Cyanidin” • Dragendorff’s Test
• Benedict’s Test Test • Wagner’s Test
• Fehling’s Test IV. Tannins and VII. Anthraquinone
II. Proteins Polyphenols Gylcosides
• Millon’s Test • Ferric Chloride Test • Borntrager’s Test
• Xanthoproteic Test V. Saponins • Modified
• Froth Test Borntrager’s Test
To determine the concentration of ethanolic crude extract from the leaves of
the Piper betle Linn. that exerted significant antibacterial activity

AGAR WELL DIFFUSION

TECHNIQUE

Medium: Mueller Hinton Agar

Bacterial Strains:
• Staphylococcus aureus
• Pseudomonas aeruginosa
To determine the concentration of ethanolic crude extract from the leaves of
the Piper betle Linn. that exerted significant antibacterial activity

Zone of inhibition Interpretation

>19mm Very Active
14mm-19mm Active
10mm-13mm Partially Active
<10mm Inactive
Compare the antibacterial activity of the ethanolic crude extract from the
leaves of the Piper betle Linn. against the reference standard, Mupirocin.

One-Way ANOVA
Two-tailed Test
To formulate and evaluate topical antibacterial ointment

0.02 g white petrolatum 6g Ointment

Microbiological Evaluation Dermal Irritation Test

• Staphylococcus aureus (IACUC approved)
• Pseudomonas aeruginosa
DETERMINATION OF PERCENTAGE YIELD

Weight of Crude extract

Percent yield = Weight of dried Piper betle leaves x 100

Percent yield = 9.92 %

ORGANOLEPTIC EVALUATION

Organoleptic
Result
Property
Color Greenish black
Odor Creosote-like odor
Appearance Syrupy consistency
 SOLUBILITY

Solvent Result
Distilled water Insoluble
Ethanol Soluble
Acetone Soluble
Ether Soluble
PHYTOCHEMICAL SCREENING

Primary Metabolites

Carbohydrates Proteins
PHYTOCHEMICAL SCREENING
Secondary Metabolites

Flavonoids Tannins Saponins Alkaloids Glycosides

TEST FOR CARBOHYDRATES
Expected Result Actual Result

Molisch’s Test Red-violet ring

Brick-red
Benedict’s Test
precipitate

Brick-red
Fehling’s Test
precipitate
TEST FOR PROTEINS
Expected Result Actual Result

Presence of flesh
Millon’s Test
color precipitate

Formation of
Xanthoproteic Test
yellow precipitate
TEST FOR FLAVONOIDS

Expected Result Actual Result

Observe colors
from orange to
Wilstatter “Cyanidin” red, crimson, and
Test magenta and
occasionally to
green or blue
TEST FOR TANNINS AND POLYPHENOLS

Expected Result Actual Result

Bluish black for
Hydrolysable
tannins
Ferric Chloride Test
Brownish green
for condensed
tannins
TEST FOR SAPONINS

Expected Result Actual Result

Formation of 1 cm
Froth Test
layer of foam
TEST FOR ALKALOIDS
Expected Result Actual Result

Formation of
Dragendorff ’s Test orange or red
precipitate

Formation of red-
Wagner’s Test
brown precipitate
TEST FOR ANTHRAQUINONE GLYCOSIDES
Expected Result Actual Result

Pink to red color

Borntrager’s Test in the ammoniacal
layers

Rose-pink color in
Modified Borntrager’s
the ammoniacal
Test
layer
TEST FOR RESINS
Expected Result Actual Result

A formation of
Phloroglucinol Test reddish-brown
color
PHYTOCHEMICAL SCREENING
No. Phytochemical Screening Result
1 Carbohydrates +
2 Proteins -
3 Flavonoids +
4 Tannins and Polyphenols +
5 Saponins -
6 Alkaloids +
7 Anthraquinone Glycosides -
8 Resins +
 Staphylococcus aureus

20%

40%
Negative Control

60%

80%
Mupirocin
 Pseudomonas aeruginosa

20%

40%
Negative
Control

60%

Mupirocin Ointment 80%

Average Zone of Inhibition in millimeters (mm)

Zone of inhibition Interpretation

>19mm Very Active
14mm-19mm Active
10mm-13mm Partially Active
<10mm Inactive
Comparison between the Ethanolic Crude Extract and the Positive Control

Zone of Inhibition (in millimeters)

Standard Standard
S. aureus P. aeruginosa
Deviation Deviation
POSITIVE 14.93 0.03 17.55 0.03
20% EXTRACT 25.85 1.13 20.63 0.58
40% EXTRACT 26.82 1.56 21.98 0.55
60% EXTRACT 27.92 1.36 22.83 0.47
80% EXTRACT 25.38 3.91 21.23 0.08

*Data expressed as mean ± SD, n=3.

Antibacterial Susceptibility Test
Dermal Irritation Test
 Staphylococcus aureus
60% Ikmo Ointment

Mupirocin

The average zone of inhibition is 23.05±1.35

mm
Negative control
 Pseudomonas aeruginosa
60% Ikmo Ointment

Mupirocin

The average zone of inhibition is 26.40±0.89

mm
Negative control
Obtained results in millimeters (mm)
Obtained results in millimeters (mm)
Dermal Irritation Test
 Dermal Irritation Test
24 h 48 h 72 h

Erythema with Eschar and Edema Erythema with Eschar Formation

B

C
Normal Skin Applied with
Simple ointment base
 Dermal Irritation Test
Day Rabbit A Rabbit B Rabbit C

Day 1

Day 7
Based on all available variables in this
study, Ikmo ointment still shows
antibacterial activity although it decreased
as compared to the Ikmo extract but it
shows higher activity in comparison with
the reference standard, Mupirocin
ointment. Furthermore, the prepared
ointment did not show any irritation to the
biological animals used in this study.
(1) Improvement of aesthetic appearance of the
prepared ointment.
(2) Test the antibacterial activity of the preparation
against Streptococcus pyogenes and Methicillin-
Resistant (MRSA) Staphylococcus aureus.
(3) Conduct in vitro drug release testing for the
prepared antibacterial ointment.
(4) Conduct stability testing for the formulated
antibacterial ointment.
(5) Evaluate the compatibility of the excipients to the
ethanolic crude extract.
(6) Conduct minimum inhibitory concentration and
minimum bactericidal concentration.
 FORMULATION OF TOPICAL ANTIBACTERIAL OINTMENT USING
THE ETHANOLIC CRUDE EXTRACT OF Piper betle LINN.
PIPERACEAE FAMILY LEAVES

DELA
CALALO, LOIS MERCED, PEREZ, EZLI QUEBEC, SHIMOMURA, VALDERAMA,
STEPHANIE B. PAOLA LAINE F. GARY JR. C. SERIE S. REGINE F.
NOREEN A.
Staphylococcus aureus
60% Ikmo Ointment

The average zone of

inhibition is 23.05±1.35 mm
Pseudomonas aeruginosa
60% Ikmo Ointment

The average zone of

inhibition is 26.40±0.89 mm