CHAPTER 1

The Problem and Its Setting

Introduction

Cases of antibiotic resistance continue to rise as it challenge our public health

sector. The resistance of bacteria to antibiotic remains unresolved problem in the world.

The use of synthetic drugs may subject the patient to a higher risk due to the unwanted

toxicity. To address this, actions must be taken, such as controlling the use of antibiotics,

developing new antibiotics, or formulating modern medication derived from herbal

plants. Traditionally used medicinal plants have constituents of known therapeutic

properties. Several plants contain flavonoids, tannins, and other polyphenolic compound

that has an antimicrobial property. The substances present in a plant can either inhibit the

growth of bacteria or kill them, with minimum toxicity to host cells. The substances from

plant which exhibit a minimum toxicity are considered candidates for developing new

antimicrobial drugs.

This study aimed to formulate topical antibacterial ointment from the ethanolic

extract from the leaves of Piper betle Linn. since there was a study that Piper betle Linn.

leaves has an antimicrobial property and shows non-toxic. Formulation of topical

antibacterial ointment using ethanolic extract from the leaves of Piper betle Linn. was

conducted with no pre formulation studies performed locally.

Background of the Study

Piper betle Linn. leaf locally known as “Ikmo”, a green, slender climbing plant,

pungent in taste, and acrid in nature. It belongs to the family Piperaceae. It is cultivated in

Sri-Lanka, India, Malaysia, East Africa, and Philippines. Piper betle Linn. has been long

used by the Filipino ancestors as an important component of their “nga-nga”. In the

Philippines, it is used together with lime and betel nut to constitute the Filipino’s

masticatory which helps in strengthening their teeth and preventing cavities and halitosis.

Nowadays, the piper betel is known for its many medicinal uses like its antioxidant,

antimalarial, cytotoxic, antifungal, antiseptic and antibacterial property.

Skin infections are caused by a wide variety of germs, and symptoms can vary

from mild to serious. Mild infections may be treated with home remedies and/or an OTC

preparations while other infections may require medical attention. There are several types

of skin infections, one of which is bacterial skin infections. Bacterial skin infections often

begin as small, red bumps that slowly increase in size. Some mild bacterial infections are

treated with topical antibiotics, but other infections are treated with oral antibiotic.

Common bacterial infections are cellulitis, boils, leprosy, and impetigo. (De Pieto and

Hiugeria, 2017)

Impetigo is a highly contagious bacterial skin infection and is one of the most

common skin infections in children. Impetigo is usually caused by one of two bacteria:

Staphylococcus aureus or Group A streptococcus. There are several ways to prevent

and/or to treat impetigo. Antibiotics are the first line treatment when multiple lesions

exist but due to the high risk of antibiotic resistant bacteria, the management of impetigo

in the future is an area of concern. There is a need to develop a new antimicrobials and
antiseptics as an alternative treatment strategy, including the new topical antimicrobials.

(Steele, 2017)

The researcher aimed to formulate a topical antibacterial ointment derived from

herbal plant with antimicrobial property against staphylococcus or streptococcus species.

Conceptual Framework

This study aims to formulate an antibacterial ointment using Ethanolic extract

from Piper betle Linn. (Ikmo). Initial screening and evaluation were conducted to

determine the primary and secondary metabolites present in the leaves. Microbiological

testing using Agar well diffusion technique was performed to evaluate the antibacterial

property of the leaves. The antibacterial activity was determined by measuring the

diameter of the zone of inhibition in millimeters using ruler and it was interpreted based

on the criteria provided. ANOVA and two-tailed t-test was conducted to test if there is

significant difference between the Ethanolic extract of Piper betle Linn. and Mupirocin

as the reference standard.

In formulating topical ointment, the extract was incorporated into the molten

simple ointment base and allowed to congeal by stirring. The formulated ointment was

packed in a collapsible tube. The formulation was evaluated to incorporate the quality to

the product. Dermal Irritation Test was performed to evaluate the safety of the product.
 Piper betle

Ethanolic
Extract

Phytochemical Microbiological Formulation

Physical Tests Tests
Screening

Odor Agar Plate Antibacterial

Flavonoids Method Sesitivity Test

Color Staphylococcus
Tannins Product
aureus Evaluation
Appearance
Saponins Pseudomonas Skin
aeruginosa Sensitivity
Solubility Test

Alkaloids

Glycosides

Carbohydrates

Proteins

Figure 1

Paradigm showing the formulation of topical antibacterial ointment using

Ethanolic extract from the leaves of Piper betle Linn.
Research Objectives

The main thrust of the study is to formulate an antibacterial ointment using ethanolic

extract from Piper betle Linn. (Ikmo) Leaves.

Specific Objectives:

Specifically, the researchers will sought to;

1. To determine the percentage yield of the ethanolic extract from the leaves of the

Piper betle Linn.

2. To evaluate the ethanolic extract from the leaves of the Piper betle Linn.

3. To determine the concentration of ethanolic extract from the leaves of the Piper

betle Linn. that exerted significant antibacterial activity in the:

3.1 Staphylococcus aureus

3.2 Pseudomonas aeruginosa

4. Compare the antibacterial activity of the ethanolic extract from the leaves of the

Piper betle Linn. against the leading antibacterial drug available in the market.

5. To formulate and evaluate topical antibacterial ointment based on the following:

5.1 Antibacterial Sensitivity Test

5.2 Product Evaluation

5.3 Dermal Irritation Test

Hypothesis

The researchers emphasize that there is no significant difference in the

antibacterial property of ethanolic extract from the leaves of Piper betle Linn. and the

standard drug, mupirocin.

Significance of the Study

This study focuses on the formulation of topical antibacterial ointment using

Ethanolic extract of Piper betle Linn. leaves. Furthermore, the results of the study will be

beneficial to the following:

To the Patients: Patient may use the formulated product derived from herbal

plant that has an antimicrobial property as an alternative drug without or lesser irritation.

To the Pharmacists: Pharmacist can use this study to furthermore investigate and

to produce new drug from new sources of natural products with antimicrobial activities

that is safe and effective.

To the Pharmaceutical Industry: The pharmaceutical manufacturers may use

this study to formulate antibacterial drugs derived from herbals as natural alternative that

is cost effective than other synthetic antibacterial drugs.

To the Physicians: Medical practitioners can prescribe alternative antibacterial

drug that is safe and effective.

To Future Researchers: This study may serve as a reference and future

researchers may do some recommendations.

Scopes and Delimitation

The study focused on the formulation of topical antibacterial ointment using

Ethanolic extract from Piper betle Linn. leaves and to evaluate its safety by conducting

Dermal Irritation Test and to evaluate its efficacy by conducting microbiological test
using agar well diffusion technique. Staphylococcus aureus and Pseudomonas

aeruginosa are the only bacteria used for the test.

The ethanolic extract was characterized by organoleptic, physical and chemical

test only. The ethanolic extract was undergone phytochemical screening of flavonoids,

tannins, saponins, alkaloids, and glycosides. The researchers did not perform all the tests

for each screenings. The researchers have only selected tests for each screenings.

The researchers used 20%, 40%, 60%, and 80% of the crude extract. The

antibacterial activity of the ethanolic extract was tested in agar plate against

Staphylococcus aureus and Pseudomonas aeruginosa using Mueller Hinton Agar as a

medium. The evaluation of ethanolic extract was done by measuring the diameter (in

millimeters) of zone of inhibition using ruler.

The concentration that exerted significant antibacterial activity was used in

formulating the topical antibacterial ointment. The researchers used polyethylene glycol

base in the formulation. The preparation was incorporated in a collapsible tube.

Antibacterial sensitivity test was performed to evaluate the antibacterial activity of

ethanolic extract of ikmo leaves in ointment. Dermal irritation test was conducted to

evaluate safety of the formulation.

Rabbits are used as test animals as its skin reaction resembles that of the human

skin, and is considered ideal and reliable in determining whether the formulated

antibacterial ointment will cause irritation or not to the skin. Albino rabbit with at least

one (1) kilogram in weight will be used in the dermal irritation test. The researchers will

use three (3) male albino rabbits to test the formulated ointment with the active ingredient
and the formulation containing the excipients only to be conducted in three (3) trials. The

rabbits will be acclimatized for seven (7) days. Twenty four (24) hours prior to testing,

their backs will be shaved approximately 6cm2 by clipping the dorsal area of the trunk

and a specific amount of antibacterial ointment from Piper betle leaves will be applied on

the area of the skin. The area will be covered with a gauzed patch. Which will be held in

place with a non-irritating tape. After four (4) hours, the patch will be detached and

residual test chemical will be removed using water.

Definition of Terms

Alkaloids. It is a basic nitrogenous organic products of plant origin, having marked

physiological actions when administered internally. They have complex, molecular

nature, consisting of nitrogen in heterocyclic rings.

Antibacterial. This is a type of antimicrobial drug used in the treatment and prevention

of bacterial infections.

Antibiotic. This is a natural or synthetic origin that have the capacity to kill or to inhibit

the growth of microorganisms.

Antibiotic resistance. It is the ability of bacteria to and microorganisms to resist the

effects of an antibiotic to which they were once sensitive.

Antifungal. Agents used in the prevention and treatment of fungal infections.

Antimalarial. A drug directed against malaria.

Antimicrobial. Inhibiting the growth of microorganisms and especially pathogenic

microorganisms.
Antioxidant. A substance that inhibits oxidation, especially one used to counteract the

deterioration of stored food products.

Antiseptic. Substances that prevent the growth of disease-causing microorganisms that

are applied to living tissue/skin to reduce the possibility of infection, sepsis, or

putrefaction.

Boil. A skin infection that starts in a hair follicle or oil gland.

Cellulitis. Bacterial infection of the skin and tissues beneath the skin.

Cytotoxic. The quality of being toxic to cells.

Herbal. Relating to or made from herbs, especially those used in medicine.

Impetigo. Is a common and highly contagious skin infection that mainly affects infants

and children.

Leprosy. A contagious disease that affects the skin, mucous membranes, and nerves,

causing discoloration and lumps on the skin and, in severe cases, disfigurement and

deformities.

Lesions. A region in an organ or tissue that has suffered damage through injury or

disease, such as a wound, ulcer, abscess, tumor, etc.

Piper betle. It is a leaf of a vine belonging to the Piperaceae family.

Polyphenolic compound. Compounds that are composed of natural, synthetic or

semisynthetic organic chemicals made up by the presence of large, multiple phenols

structure.
Synthetic. Made by chemical synthesis, especially to imitate a natural product.

Topical. A route of administration in which the site of drug intake is located beneath the

skin.

Toxicity. The quality of being toxic or onous.