1

CHAPTER 1

The Problem and Its Setting

Introduction

In a bulletin published by World Health Organization in 2011 noted that

antimicrobial pipeline is drying-up and cases of antibiotic resistance continue to increase

therefore challenging local and international public health sector. The resistance of

pathogens 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 responsible use of antibiotics, developing new

antibiotics, or formulating plant-derived pharmaceuticals. 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 metabolites from plant which exhibit a

minimum toxicity are considered candidates for developing new antimicrobial drugs.

This study aimed to prepare topical antibacterial ointment from the ethanolic

crude extract from the leaves of Piper betle Linn. locally known as Ikmo since there was

a study that Ikmo leaves have an antimicrobial property and shows no toxicity.

Preparation of topical antibacterial ointment using ethanolic crude extract from the leaves

of Ikmo was conducted with no previous studies of formulating ointment locally.

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Background of the Study

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 over-

the-counter 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)

Ikmo leaves is 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. Ikmo leaves 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
 3

its many medicinal uses like its antioxidant, antimalarial, cytotoxic, antifungal, antiseptic

and antibacterial property.

The researcher aimed to prepare a topical antibacterial ointment derived from

herbal plant with antimicrobial property against staphylococcus or streptococcus species.

Conceptual Framework

Preparation of topical antibacterial ointment using the ethanolic crude extract of

(Piper betle linn. Piperaceae family) Ikmo leaves.

This study aims to prepare an antibacterial ointment using Ethanolic crude extract

from Ikmo. Cases of antibiotic resistance in the world is continuously rising and

resistance of bacteria to antibiotic remains an unresolved problem. Skin infections 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. Antibiotics are

usually the first line treatment when it comes to skin infections but due to the high risk of

antibiotic resistant bacteria, the management of skin infections in the future is an area of

concern therefore, the development of a new antimicrobials and antiseptics as an

alternative treatment strategy is a need, including the new topical antimicrobials.

Ikmo is a herbal plant that is known to have an antimicrobial activity against

bacteria especially staphylococcus or streptococcus species that’s why the researchers

conducted the study, Preparation of topical antibacterial ointment using ethanolic crude

extract from the leaves of Ikmo with no previous studies of formulating ointment locally.
 4

Piper betle

Ethanolic
Extract

Phytochemical Antibacterial Preparation

Physical Tests Susceptibility Tests
Screening

Agar Plate Method Antibacterial

Primary Odor
metabolites (Dose Susceptibility
determination) Test

Secondary Color
Staphylococcus Dermal Irritation
metabolites aureus Test

Appearance

Pseudomonas
aeruginosa
Solubility

Figure 1

Paradigm showing the preparation of topical antibacterial ointment using

Ethanolic crude extract from the leaves of Ikmo.
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Research Objectives

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

extract from Ikmo Leaves.

Specific Objectives:

Specifically, the researchers will seek to:

1. To obtain ethanolic crude extract from the leaves of Ikmo and determine the

percentage yield;

2. Evaluate the ethanolic leaf extract using organoleptic, solubility and chemical test

(determination of metabolites);

3. 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 Irritation Test.

5. Compare the 60% ethanolic crude extract of Ikmo leaves against Ikmo ointment.

Hypothesis

There is significant difference between the prepared ointment with ethanolic

crude Ikmo leaf extract and the standard drug Mupirocin ointment. (p>0.05).
 6

Significance of the Study

Antimicrobial resistance continues to post threat to global civilization and the

World Health Organization urges the public to search for new antimicrobial agents

therefore this study focuses on the preparation of topical antibacterial ointment using the

ethanolic crude extract of Ikmo leaves. Furthermore, the results of the study will give

significance to the following:

To the Patients: Patient may use the prepared product derived from herbal plant

as an alternative antimicrobial drug without or lesser irritation.

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

produce new drug from natural source that has antimicrobial activities which is safe and

effective.

To the Pharmaceutical Industry: The pharmaceutical manufacturers may use

this study to prepare antibacterial drugs derived from natural source that is cost effective

than other synthetic antibacterial drugs.

To the Physicians: Medical practitioners can prescribe herbal plants as

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 provided.

Scopes and Delimitation

The study focused on the preparation of topical antibacterial ointment using

ethanolic crude extract of Ikmo leaves.

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The ethanolic crude extract of Ikmo was characterized by organoleptic, physical

and chemical test only. The ethanolic crude extract was undergone phytochemical

screening of carbohydrates, proteins, flavonoids, tannins, saponins, alkaloids, and

anthraquinone glycosides, and resins only. The researchers did not perform all the tests

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

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

ethanolic crude extract of Ikmo undergone microbiological screening against

Staphylococcus aureus and Pseudomonas aeruginosa using Mueller Hinton Agar as a

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

(in millimeters) of zone of inhibition using Vernier caliper. The zone of inhibition of

ethanolic crude extract of Ikmo against Staphylococcus aureus and Pseudomonas

aeruginosa was interpreted based on the table provided from the manual (Source:

Limuaco O.M, et.al., 2014).

The researchers used the concentration that exhibited the greatest zone of

inhibition for the preparation of the ointment. The researchers used 60% ethanolic crude

extract of Ikmo and was subjected to prepare an ointment and was compared to

mupirocin ointment to see if the prepared ointment still have the activity against

microorganism. The preparation was packed in collapsible tube and was properly labeled.

The preparation undergone dermal irritation test to evaluate its safety using rabbits as an

animal model. All six albino rabbits were examined for signs of erythema and oedema

based on the grading of skin reactions provided from OECD/OCDE.

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Definition of Terms

Alkaloids. It is a basic nitrogenous organic products of plant origin with important

physiological effects in humans.

Antibacterial. Anything that kills bacteria or suppresses their growth or their ability to

reproduce. An agent that kills or suppresses the ability of bacteria to grow and reproduce.

Antibiotic. A natural or synthetic origin that is used to treat bacterial infections.

Antibiotic resistance. The ability of bacteria and other microorganisms to resist the

effects of an antibiotic to which they were once sensitive.

Antifungal. An agent used in the prevention and treatment of fungal infections.

Antimalarial. An agent used to prevent or cure malaria.

Antimicrobial. An agent used to destroy or inhibit the growth of microorganisms

especially pathogenic microorganisms.

Antioxidant. A chemical compound that protects cells against the effects of free radicals.

Antiseptic. A substance that inhibits the growth or action of microorganisms especially

in living tissue.

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

Cellulitis. A spreading bacterial infection of the skin and tissues characterized by

redness, warmth, swelling, and pain underneath the skin.

Cytotoxic. Any agent or process that kills cells.

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

 9

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

children.

Leprosy. A contagious disease caused by that affects the skin and peripheral nerves and

characterized by the formation of nodules or macules that enlarged and spread

accompanied by loss of sensation with eventual paralysis, wasting of muscle, and

production of 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. A leaf of a vine belonging to the Piperaceae family.

Polyphenolic compound. A compound composed of natural, synthetic or semisynthetic

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

Synthetic. A substance 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 poisonous.

Volatile oil. A concentrated hydrophobic liquid containing volatile aroma compounds

from plants.
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Chapter 2

Review of related literature and studies

This chapter covers the compiled local and foreign literature and studies. This

chapter aims to present the information that the researchers were able to gather for better

understanding of the readers.

In the past years, the use of medicinal plants, from different parts of the world, for

their antimicrobial property has been increasingly reported. According to the reports, the

plant extracts show a different target site other than those targeted by the synthetic

antibiotics and that these plant extracts targeting other sites with be effective against the

drug resistant microbial pathogens.

The modern era of antibiotics started with the discovery of Penicillin by Sir

Alexander Fleming in 1928. Since then, Antibiotics have transformed modern medicine

and saved millions of lives. Antibiotics were first prescribed to treat serious infections in

the 1940s. Antibiotics have not only saved patients’ lives, they have played a pivotal role

in achieving major advances in medicine and surgery. They have successfully prevented

or treated infections that can occur in patients who are receiving chemotherapy

treatments; who have chronic diseases such as diabetes, end-stage renal disease, or

rheumatoid arthritis; or who have had complex surgeries such as organ transplants, joint

replacements, or cardiac surgery. The rapid emergence of resistant bacteria is occurring

worldwide, endangering the efficacy of antibiotics, which have transformed medicine and

saved millions of lives. Many decades after the first patients were treated with antibiotics,

bacterial infections have again become a threat. The antibiotic resistance crisis has been

attributed to the overuse and misuse of these medications, as well as a lack of new drug
 11

development by the Pharmaceutical Industry due to reduced economic incentives and

challenging regulatory requirements. Penicillin was successful in controlling bacterial

infections among World War II soldiers. However, shortly thereafter, Penicillin

resistance became a substantial clinical problem, so that, by the 1950s, many of the

advances of the prior decade were threatened. The overuse of antibiotics clearly drives

the evolution of resistance. Epidemiological studies have demonstrated a direct

relationship between antibiotic consumption and the emergence and dissemination of

resistant bacteria strains. In bacteria, genes can be inherited from relatives or can be

acquired from nonrelatives on mobile genetic elements such as plasmids. This horizontal

gene transfer (HGT) can allow antibiotic resistance to be transferred among different

species of bacteria. Resistance can also occur spontaneously through mutation.

Antibiotics remove drug-sensitive competitors, leaving resistant bacteria behind to

reproduce as a result of natural selection. (Ventola, 2015)

The infectious disease society of america has considered the following bacteria as

especially challenging in terms of management: Methicillin-resistant staphylococcus

aureus (MRSA), Vancomycin-resistant enterococcus (VRE), extended spectrum beta

lactamase (ESBL)-producing and Carbapenem-resistant enterobacteriaceae (CRE),

Metallo-b-lactamase (MBL)-producing Pseudomonas aeruginosa and Acinetobacter

baumannii. (D. L. Valle Jr. Et. Al., 2015)

The same emerging MDR bacteria are also an immense threat in Asia. There have

been reports about the occurrence of ESBL-producing enterobacteriaceae in imported

fresh culinary herbs from Thailand, Vietnam, and Malaysia. These culinary herbs are

usually consumed without proper heating. Acinetobacter baumannii, being able to exhibit
 12

a wide spectrum of antimicrobial resistance mechanism, has also emerged as one of the

most problematic bacteria.

The current problem with the MDR bacteria is a serious, global medical crisis

which requires constant monitoring. Scientists are seeking for more natural or organic

materials as a solution for the diminishing efficacy and increasing toxicity of synthetic

drugs which further aggravate the problem. Traditional medicinal plants are being studied

further for the purpose of finding solution to the emerging problem regarding MDR

bacteria.

Bacteria was one of the most common cause of deadly diseases but with the

discovery of antibiotics, this problem was slowly resolved. However, due to inadequate

use of antibiotics, multidrug resistance developed and continues to be a challenge to the

healthcare sector.

Multidrug resistance is often described as decreased in vitro biological activity to

multiple class of drugs without considering remaining effective therapies. This leads to

bacterial strains being resistant to antibiotics. Some of the bacterial strains that are

resistant to multiple drugs are Staphylococcus aureus, Pseudomonas aeruginosa.

Staphylococcus aureus is a golden spherical bacterium frequently found in the

nose, throat, intestine, vagina, and skin of human body. It is a round and bunched

together pathogen and has the ability to cause different kinds of mild to severe infections

and it is capable to adapt fast to the different environmental conditions.

Pseudomonas aeruginosa is the most common pathogen isolated from patients

who have been hospitalized longer than 1 week, frequently causing nosocomial

infections. Some strains of MDR P. aeruginosa have been found to be resistant to nearly
 13

all antibiotics, including aminoglycosides, cephalosporins, fluoroquinolones, and

carbapenems.

Drugs intended for skin infections are usually formulated into ointments for

topical application. Ointments are greasy and they stay on the surface of the skin and are

not well absorbed, thus are “occlusive”. They are best used on dry skin and they trap

moisture that’s why they are able to keep the skin moist for longer periods of time. They

have lesser chance of causing allergy because of few preservatives. Ointments allow

better penetration of the active ingredient in the topical medication and they are better

used on sensitive skin.

Some of the available topical antibacterial medicines in the Philippines includes

Mupirocin ointment (Mupicin®, Bactroban®), Silver sulfadiazine (Silvex®,

Flammazine®), and Sodium fusidate ( Woncare® ).

There are limited topical antibacterial medicines available in the Philippines

because skin and soft tissue infections such as scrapes and scratches or mild folliculitis,

do not usually require antibiotic treatment. In primary care, management should focus on

good skin hygiene, for patients with infected eczema, for wound management, or for

other skin infections, topical antibacterial medicines are not usually prescribed or they

may only be appropriate as second line option.

Ikmo from the family of Piperaceae is a dioecious, shade-loving, perennial

evergreen climbing vine whose height reaches 2 to 4 meters. Ikmo leaves, which has a

glossy heart-shaped physical appearance, is very popular in the Philippines where it is

used as a masticatory with nga-nga (Areca catechu) and apog (lime).

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Ikmo is best grown in tropical areas like Philippines, Malaysia, Sri Lanka and

Indonesia. In the Philippines, it is best grown in region VI, in provinces like Ilo-Ilo,

Bacolod, and Cordillera Mountains. Its production is a very tedious process and it is not

cultivated on a large-scale basis because it is not that popular.

The Ikmo leaf has been described to have piperol-a, piperol-b, methyl piper

betlol. The Ikmo leaves have starch, sugars, diastases and an essential oil composing of

terpinen-4-ol, safrole, allyl pyrocatechol monoacetate, eugenol, eugenyl acetate, hydroxyl

chavicol , eugenol , piper betol and the betle oil contains cadinene carvacrol, allyl

catechol, chavicol, p-cymene, caryophyllene, chavibetol, cineole, estragol, etc. as the key

components. (Dwivedi, Et. Al., 2014)

Present constituents Potential antibacterial activity

Chavibetol None

Eugenol Yes

Hydroxychavicol Yes

Allypyrocathecol Yes

Quercetin Yes

Β-caryophyllene None
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Chavibetol is a natural chemical compound of the phenylpropanoid class. It is the

most important component of the essential oil from the leaves of the Ikmo plant. It is

an aromatic compound with a spicy odor and is an isomer of eugenol. (Dwivedi, Et. Al.,

2014)

Eugenol, one of the principal constituent of Ikmo leaf, has also been shown to

possess anti-inflammatory property in a variety of animal models of studies with various

inflamogens. Antimicrobial, analgesic, anti-oxidant, antiviral and anticancer activity,

other identified activities such as its anti-ulcerogenic potential and effect on osteoporosis

and especially its effect on the Central Nervous System (CNS) encompassing seizure

control, Parkinson’s disease, antidepressant effects etc. (Dwivedi, Et. Al., 2014)

The new, immature leaves contains various beneficial bio- active compounds,

among which hydroxychavicol is most important phenolic compound which reported to

possesses anti carcinogenic, anti nitrosation, anti-mutagenic effects beside this, it has a

considerable potency to act as an anti- inflammatory, antioxidant, antibacterial, anti-

platelet and anti- thrombotic effects without impairing haemostatic function. In the

aqueous extract of Ikmo leaf it is reported to exhibit useful bioactivities – antimutagenic

and anticarcinogenic activities, whereas isolated from the chloroform withdrawal from

aqueous extract of Ikmo leaves show inhibitory action alongside oral cavity pathogens.

0.5% hydroxychavicol inhibited the biofilm produced by anaerobes and biofilm produced

in pooled saliva the use of hydroxychavicol as an oral care agent. Hydroxychavicol show

compelling anti- inflammatory action by considerably inhibits the phrase of the pro

inflammatory cytokine TNF-α. Methyl chavicol, a biogenic oxygenated aromatic

compound, reported to have antioxidant activity. (Dwivedi, Et. Al., 2014)

 16

The phenolic constituent allyl pyrocatechol obtained from the leaves, show action

against obligate oral anaerobes responsible for halitosis. The leaf extract also has a

stimulatory outcome on pancreatic lipase and antioxidant activity. Oral administration of

allyl pyrocatechol at different doses accelerates the rate of remedial of gastric lesion

induced by indomethacin due to its antioxidative and mucin defensive properties.

(Dwivedi, Et. Al., 2014)

Quercetin is one of the most important dietary flavonoids belong to a group of

flavonols. It occurs chiefly as glycosides, but other derivatives of quercetin have been

recognized as well. Joined substituent’s changing the biochemical activity and

bioavailability of molecules when compare to the aglycone. Quercetin has also been

verified to exhibit the antiviral, antibacterial, anticarcinogenic and anti- inflammatory

properties. The anticarcinogenic property of quercetin result from its important impact on

an increase in the apoptosis of mutated cells, inhibition of DNA synthesis, inhibition of

cancerous cell growth, decrease and alteration of cellular signal transduction pathways.

Animal evidence suggest quercetin’s antioxidant effects provides protection of the brain,

heart, and other tissues adjacent to ischemic a- reperfusion injury, toxic compounds, and

other factors that can persuade oxidative stress. Β-caryophyllene is a chief volatile

compound establish in huge amounts in different spice and food plants. (Dwivedi, Et. Al.,

2014)

β-caryophyllene has shown to possess potent anti-inflammatory properties. Β-

caryophyllene is an fda- approved food additive and it is apparently a non-toxic

compound with no genotoxic or cytotoxic effect in vivo. Clinical studies prove its

efficiency in treating endometriosis. Β-caryophyllene exerts anti- inflammatory activity

 17

by acting as a potent, selective and non- psychoactive full agonist for cb2 receptor in

vivo. (Dwivedi, Et. Al., 2014)

Through the discovery of antibiotics, millions of lives were saved but because of

the overuse and misuse of these medications, antibiotic resistance arose which is now,

again, endangering millions of lives. Multidrug resistance leads to strains of bacteria

being resistant to antibiotics. The current problem with the multidrug resistant bacteria is

a serious, medical crisis which requires constant monitoring. Some of the bacteria that are

resistant to multiple drugs are staphylococcus aureus and pseudomonas aeruginosa.

Because of the emergence of antibiotic resistance, scientists are striving to discover a

more natural or organic source to be able to synthesize a drug that will solve the problem

associated with synthetically made drugs. In line with seeking for natural sources, various

studies about herbal plants are being conducted. One of the plants that have been used

since the early days is the piper betel. Piper betel, from the family Piperaceae, contains

constituents such as eugenol, hydroxychavicol, allyl pyrocatechol that has potential

antibacterial activity. Thus, piper betel may be used in formulating a medicine against

certain bacterial strain.

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CHAPTER 3

Methods and Procedures

This chapter is concerned with the research methodology, which covers the

procedures used in the preparation of a topical antibacterial ointment using Ethanolic

Crude Extract from the leaves of Ikmo.

Method of Research Used

The researchers used the experimental method of research to be able to prepare a

topical antibacterial ointment from the ethanolic crude extract of Ikmo Leaves.

1. Determination of percentage yield ethanolic crude extract from the leaves of

Ikmo.

The fresh Ikmo leaves were collected in the province of Nueva Ecija. The leaves

were washed with running tapped water, air- dried for 1 week, then grinded into powder

using blender. 500 g of Ikmo leaf powder in 2500 mL of Ethanol were mixed and

macerated for 48 hours. The mixture was subjected to filtration, then the filtrate was

collected and was incipient dried. The percentage yield was able to determine using the

given formula below.

Percentage Yield = Weight of Ethanolic Crude Extract

___________________________ X 100
Weight of dried Ikmo leaves

2. Evaluation of ethanolic crude extract of Ikmo leaves

2.1 Organoleptic Evaluation

In a trace amount of ethanolic extract, the odor, color, and physical appearance

was observed and results were recorded.

2.2 Physical Test

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Put trace amount of ethanolic crude extract in each test tube containing alcohol,

water, acetone, and ether, respectively to determine the solubility of the extract.

2.3. Chemical Test

2.3.1 Determination of the presence of Primary Metabolites

2.3.1.1 Test for Carbohydrates

The ethanolic crude extract, about 2 grams equivalent, was evaporated and the

residue was taken then mixed with 10 mL distilled water and divided into nine equal

parts:

2.3.1.1.1 Molisch’s Test

2 mL of the prepared filtrate was mixed with 0.2 mL of 10% α-naphthol

which was added to 2 mL of sulfuric acid. Presence of bluish violet zone indicates

a positive result.

2.3.1.1.2. Benedict’s Test

5mL of Benedict’s reagent was added to 1mL of the filtrate, then heat the

mixture. Reducing sugars are present if there is formation of red precipitate.

2.3.1.1.3 Fehling’s Test

Boil about 5 mL of freshly prepared Fehling’s solution (2.5 mL each of

Fehling’s A & B) using test tube. Add an equal quantity of plant extract and boil

again. A brick red precipitate indicates the presence of reducing sugars. (Limuaco

O.M, et.al., 2012)

2.3.1.2 Test for Proteins

Evaporate 2 g equivalent of the plant extract. Take up the residue with 10

mL of water. Perform the following tests: (Limuaco O.M, et.al., 2012)

 20

2.3.1.2.1. Millon’s Test

Add to 1 mL of the aqueous extract, 10 drops of Millon’s reagent and

place in a boiling water bath. Presence of flesh color indicates the presence of

proteins. (Limuaco O.M, et.al., 2012)

2.3.1.2.2Xanthoproteic Test

Add 1 mL of the aqueous extract, 10 drops of nitric acid. Formation of

yellow precipitate indicates the presence of proteins. (Limuaco O.M, et.al., 2012)

2.3.2 Determination of the presence of Secondary Metabolites

2.3.2.1 Test for Flavonoids

0.5 mg of magnesium turnings and 5 drops of concentrated HCl was added

dropwise to 1 mL of ethanolic extract. A pink, scarlet, crimson red or occasionally

green to blue color standing for 3 minutes indicates the presence of flavonoids.

(Limuaco O.M, et.al., 2012)

2.3.2.2 Test for Tannins

A few drops of 5% ferric chloride solution was added to 1-2 mL of the

ethanolic extract. A green or brown color indicates the presence of tannins.

(Limuaco O.M, et.al., 2012)

2.3.2.3 Test for Saponins

2.3.2.3.1 Froth Test

1mL of ethanolic extract was added with 10 mL distilled water in a test

tube. The solution was shaken vigorously for 30 seconds and allow to stand for 10

minutes. Observed for “honeycomb” froth. Froth with greater than 2cm height
 21

from the surface of the liquid persists in 10 minutes indicates the sample positive

for saponins. (Limuaco O.M, et.al., 2012)

2.3.2.4 Test for Alkaloids

2.3.2.4.1. Dragendorff’s Test

5 ml of distilled water was added to 2mL of ethanolic extract, then 2M

HCl was added until an acid reaction occur. 1 ml of Dragendorff’s reagent was

then added to the mixture. Formation of orange or orange red precipitate indicated

the presence of alkaloids. (Limuaco O.M, et.al., 2012)

2.3.2.4.2. Wagner’s Test

2 mL of the ethanolic crude extract was acidified with 1.5% v/v of HCl. A

few drops of Wagner’s reagent were added to the acidified extract. A formation of

red-brown precipitate indicates the presence of alkaloids. (Limuaco O.M, et.al.,

2012)

2.3.2.5 Test for Glycosides

2.3.2.5.1. Borntrager’s Test

Take an equivalent of 1g sample from the prepared stock extract and

evaporate to incipient dryness over steam bath. Take the residue with 10 mL

distilled water, filter and discard the residue; extract the aqueous filtrate with 5

mL portions of benzene twice and combine and combine two portions of benzene

extracts. Divide combined benzene extracts into two portions. One portion serves

as the control. Treat the other portion with 5 mL ammonia solution and shake.

Compare the result with the control. A red coloration in the ammoniacal layers

indicates the presence of anthraquinones. (Limuaco O.M, et.al., 2012)

 22

2.3.2.5.2. Modified Borntrager’s Test

Take an equivalent 1g sample from the prepared stock extract and

evaporate to incipient dryness over a steam bath. Add 10 mL 0.5M KOH and 1

mL of 5% H2O2 and stir. Heat the resulting mixture over steam bath for 10

minutes. Filter and discard the residue. Acidify filtrate with glacial acetic acid.

Collect the filtrate and extract it with two 5mL portions of benzene; combine with

benzene extract and divide it in two portions, one portion will serve as the control

and alkalinify the other portion with ammonia and shake. Compare the result with

the control. A pink color indicates a positive result. (Limuaco O.M, et.al., 2012)

3. Microbiological Evaluation of Ethanolic crude extract

3.1 Preparation of Mueller Hinton Agar as a Culture media

38.0 grams of the medium were suspended in 1000 mL of distilled water. Heat to

boiling to dissolve the medium completely. The medium was sterilized using an

autoclave at 121°C for 15 minutes, then cooled to 45 to 50°C and poured into sterile petri

plates.

3.2 Preparation of Inoculum

Staphylococcus aureus and Pseudomonas aeruginosa are bacteria used in the

study. The inocula used were undergone confirmatory test using agar plates to determine

its purity. Nutrient agar was used to test the purity of Pseudomonas aeruginosa, and on

the other hand, Mannitol Salt Agar was used for Staphylococcus aureus. The researchers

prepared McFarland standard as a reference to adjust the turbidity of bacterial

suspensions so that the number of bacteria will be within a given range to standardize

microbial testing.
 23

3.2.1 Inoculation

The researchers used plated medium technique; made a zigzag streak on the

surface of the plate using the sterile cotton swab.

3.2.2 Preparation of Control

Mupirocin ointment was used as a reference standard because Mupirocin ointment

was the most recommended topical antibiotic and reserved for treating MRSA infection.

3.3 Microbiological Testing

Different concentrations of ethanolic crude extract of Ikmo. (20%, 40%, 60%,

80%) and, the water and tween 80 as the negative control were placed on the agar plate

individually and covered aseptically. The plates were incubated at 35-37°C for 16-18

hours. The antibacterial activity was determined by measuring the diameter of the zone of

inhibition in millimeters using a vernier caliper. The result was interpreted based on the

table provided.

TABLE 2

Diameter of Zone of
Activity Interpretation
Inhibition (mm)

< 10 Inactive

10 – 13 Partially active Resistant

14 – 19 Active Intermediate

> 19 Very active Susceptible

Source: Limuac,o O.M. et.al., 2014

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4. Preparation and Evaluation of Topical Antibacterial Ointment

The study aims to prepare and evaluate an antibacterial topical ointment based on

the following:

4.1.1 Preparation of Ointment by Incorporation

The extract was incorporated into the molten simple ointment base and allowed to

congeal by stirring. The preparation was packed in collapsible tube.

Preparation of Ingredients

Preparation of White Ointment (based on USP-NF)

White wax………………………………. 2.00 grams

White Petrolatum q.s ad………………… 1000 grams

Source: Limuaco, O.M. et.al. 2013

Modification in the Preparation of the White Ointment using the Ethanolic Crude

Extract of Ikmo

Ethanolic Crude Extract of Ikmo ………. 6.00 grams

White wax ……………………………… 0.02 grams

White Petrolatum q.s. ad………………... 10.00 grams

COMPOUNDING PROCEDURES

1. Weigh all the ingredients accurately.

2. Melt the white wax in a suitable dish on a water bath.

3. Add white petrolatum, warm until liquefied. Discontinue heating.

4. Incorporate the extract into the molten simple ointment base and stir the mixture until

it begins to congeal.
 25

5. Place in an appropriate container.

6. Label the container properly.

Source: Limuaco, O.M. et.al. 2013

4.1.2 Dermal Irritation Test

The researchers used six (6) male albino rabbits to test the prepared ointment with

the active ingredient and the preparation containing the excipients only to be conducted

in three (3) trials. The rabbits were acclimatized for seven (7) days. Twenty four (24)

hours prior to testing, their backs was shaved and a pinch of the preparation was applied

on the shaved area and covered with gauzed patch. The animals should be observed up to

14 days after removal of the patches. The results were examined for signs of erythema

and oedema based on the grading of skin reactions provided from OECD/OCD and the

responses scored at 60 minutes, and then at 24, 48 and 72 hours after patch removal.
 26

5. Statistical Analysis

Statistical analysis was conducted to test if there is significant difference between

the Ikmo ethanolic crude extract and Mupirocin ointment as the positive control.

4.1. One-way ANOVA

One-way ANOVA was used to determine if there is a significant

difference between the prepared ointment and the positive control, Mupirocin ointment.

4.2. Two-tailed T-Test

Two-tailed T-test was used to test if there is a significant difference

between the average zones of inhibition of the Ikmo ethanolic crude extract and the

positive control, Mupirocin ointment.

 27

Piper betle

Ethanolic
Crude
Extract

Phytochemical Antibacterial Preparation

Screening Physical Tests Susceptibility Tests
 Flavonoids  Odor  Staphylococcus  Antibacterial
 Tannins aureus Susceptibility
 Color
Test
 Saponins  Appearance  Pseudomonas  Dermal
 Alkaloids  Solubility aeruginosa Irritation Test
 Glycosides
 Resins
 Carbohydrates
 Proteins

Figure 1

Paradigm showing the preparation of topical antibacterial ointment using

Ethanolic Crude extract from the leaves of Ikmo
 28

Chapter 4

Presentation, Analysis, and Interpretation of Data

This chapter provides the presentation, analysis and interpretation of the data

gathered from the tests conducted to the ethanolic crude extract from the leaves of Piper

betle Linn.

1. Percentage Yield of Ethanolic Crude extract from the leaves of Piper betle Linn.

The Ethanolic Crude Extract from Piper betle Linn. were obtained by maceration

using 95% ethanol as solvent then was evaporated to incipient dryness. The ethanolic

crude extract was weighed and the percentage yield was computed. The percentage

computed was 9.9222%.

2. Evaluation of ethanolic crude extract of Piper betle Linn. leaves

2.1 Organoleptic Tests Results

TABLE 1

Organoleptic Tests Results

ORGANOLEPTIC EVALUATION INTERPRETATION

Color Greenish black Due to the presence of plant

pigments and other organic

constituents (Douma, M., 2008))

Odor Creosote-like odor Due to the presence of aromatic

compounds and the application of

heat (Evans, 2010)

 29

Physical Syrupy consistency Syrupy consistency due to the

appearance presence of resin compounds

(Evdokimor, 2014)

2.2 Results for Solubility Test of the Ethanolic Crude Extract

The ethanolic crude extract was soluble in acetone, ethanol, and ether and

insoluble in water (USP Reference table for solubility)

TABLE 2

Results for Solubility Test of the Ethanolic Crude Extract

Solvent Relative Solubility Interpretation

Water Insoluble Due to presence of resinous

material which is insoluble

in water.

Acetone Soluble Presence of organic

substance

Ethanol Soluble Presence of ethanol-soluble

constituents

Ether Soluble Presence of non-polar

organic metabolites
 30

2.3 Chemical Test of the Ethanolic Crude Extract

TABLE 3

Results of Chemical Test

Determination of the presence of primary metabolites

Expected result Actual result Discussion

Test for Carbohydrates

Molisch’s Test Red-violet ring Presence of Presence of

red-violet ring Carbohydrates

Brick red Presence of

Formation of brick- Carbohydrates,
red precipitate reducing
Benedict’s Test precipitate
sugars

Brick-red
Formation of brick red Presence of
Fehling’s Test precipitate precipitate Carbohydrates,
reducing
sugars
 31

Test for Proteins

Presence of flesh Absence of

Millon’s Test No Formation of flesh
proteins,
color precipitate color
specifically
tyrosine

Absence of
Xanthoproteic Formation of No Formation of yellow proteins,
color specifically
Test yellow precipitate aromatic
amino acids

For the primary metabolites, the ethanolic crude extract of Ikmo leaves contains

carbohydrates, specifically reducing sugars, while it can be noted that proteins are not

detected primarily due to the application of heat during the preparation of crude extract

since proteins easily denatured when heat is applied.

 32

Determination of the presence of secondary metabolites

Expected result Actual result

Test for Flavonoids

colors from orange

Presence of
Wilstatter to red, crimson, benzo-pyrone
Crimson red which is a
“Cyanidin” and magenta and common
nucleus
Test occasionally to present on
flavonoids
green or blue

Test for Tannins and Polyphenols

Bluish black for Presence of

conjugated
Ferric Chloride hydrolysable double bond
ring which is
Test tannins Greenish black commonly
present on
Brownish green condensed or
non-
for condensed hydrolysable
tannins
tannins

Test for Saponins

Froth Test Formation of 1 cm

No formation of 1cm Absence of
layer of foam layer foam Saponins
 33

Test for Alkaloids

Dragendorff’s Formation of Formation of red/orange Presence of

precipitate Alkaloids
Test orange or red

precipitate

Wagner’s Test Formation of red- Presence of

Formation of red brown
precipitate Alkaloids
brown precipitate

Test for Anthraquinone Glycosides

Borntrager’s Pink to red color in

Absence of pink to red Absence of
Test the ammoniacal color in the ammoniacal anthraquinone
layer nucleus
layers

Modified Rose-pink color in

Absence of rose-pink Absence of
Borntrager’s the ammoniacal color in the ammoniacal anthraquinone
layer nucleus
Test layer
 34

Test for Resins

A formation of
Phloroglucinol
reddish-brown Reddish-brown color Presence of Resins
Test
color

The ethanolic Ikmo leaf extract contains polyphenols like flavonoids, tannins and

presence of alkaloids, and resins.

3. Determination of the antibacterial property of the ethanolic crude extract from

the leaves of Piper betle Linn.

Four different concentrations (20%, 40%, 60%, and 80%) of ethanolic crude

extract of Ikmo have the capability of inhibiting both Staphylococcus aureus and

Pseudomonas aeruginosa.

The 20% extract (25.83±1.13mm), 40% extract (26.82±1.56mm), 60% extract

(27.92±1.36mm), 80% extract (25.38±3.91mm) were very active against Staphylococcus

aureus.

The 20% extract (20.63±0.58mm), 40% extract (21.98±0.55mm), 60% extract

(22.83±0.47mm), 80% extract (21.23±0.08mm) were very active against Pseudomonas

aeruginosa.

4. Preparation and Evaluation of Topical Antibacterial Ointment

The preparation was prepared by incorporation method. 60% of the ethanolic

crude extract of Ikmo was used as an active ingredient and white ointment was used as an

ointment base. The extract, white wax, and white petrolatum were mixed together and
 35

allowed to congeal by stirring. The preparation was packed in collapsible tube and

labeled according to its basic guidelines, and stored in a cool, dry place.

The preparation was undergone antibacterial susceptibility test and was compared

to Mupirocin ointment. The 60% Ikmo ointment has the capability of inhibiting

Staphylococcus aureus and Pseudomonas aeruginosa with an average zone of inhibition

of 23.05±1.35mm and 26.40±0.89mm respectively and was comparable to Mupirocin

ointment (17.55±0.03mm).

Results of Dermal Irritation Test

Rationale for in vivo testing:

A dose of 0.5 g of the preparation was applied to a small area (approximately 6
cm2) of hair-free skin and covered with a gauze patch, which is held in place with non-
irritating tape. At the end of the exposure period, which is normally 4 hours, residual test
chemical was removed, using water. The rabbits was expected to observe up to 14 days
after removal of the patches. The experiment was terminated in the 7th day since the hair
skin of the rabbit started to grow. All six albino rabbits were examined for signs of
erythema and oedema according to the grading of skin reactions from OECD, and the
responses scored at 60 minutes, and then at 24, 48 and 72 hours after patch removal.

Test chemical:
60% Ikmo Leaf Extract Ointment

Vehicle:
White Petrolatum

Test animals:
(6) Male albino rabbits from Department of Science and Technology (DOST) with
weight of NLT 1.5 kg were used.

Test condition:
The patch was loosely held in contact with the skin by means of a suitable semi-occlusive
dressing. Access by the animal to the patch and ingestion or inhalation of the test
chemical was prevented.
 36

Results:
24 h 48 h 72 h

Erythema with Erythema with

Eschar and Eschar Formation
Edema
B

C
Applied with
Normal Skin Simple ointment
base

Rabbits A, B, and C were examined for signs of erythema and oedema and graded
according to the grading of skin reactions from OECD. Rabbits A, B, and C have seen no
erythema and oedema formation.

5. Statistical Analysis: Comparison between the Ethanolic Crude Extract and the

Ikmo ointment
 37

Comparison between the Ikmo ointment and the Mupirocin ointment

 38

CHAPTER 5

Summary of Findings, Conclusion and Recommendation

This chapter contains the summary of findings from the performed tests, the

conclusions made and the recommendations.

Summary of Findings

Perfectly mature Piper betle, L. leaves were collected, dried and macerated using

95% ethanol and subjected to reflux distillation. The extractives were dried to incipient

dryness and the percentage yield of the extract was 9.9222%. The Ikmo leaves extract is

greenish-black, with distinct creosote-like odor and has syrupy consistency. The ethanolic

crude extract of Piper betle Linn. Leaves (Ikmo) contains reducing sugars, flavonoids,

tannins, alkaloids, and resins. The microbiological assay was conducted using agar-plate

method using two (2) test organisms, namely S. aureus and P. aeruginosa. The assay was

done in three trials.

Increasing dose of the ethanolic crude extract (increments of 20%) of Ikmo were

used and exhibited an average zone of inhibition of 25.85±1.13 & 20.63±0.58 mm,

26.82±1.56 & 21.98±0.55 mm, 27.92±1.36 & 22.83±0.47 mm and 25.38±3.91 &

21.23±0.08 mm against Staphylococcus aureus and Pseudomonas aeruginosa,

respectively. The 60% concentration was used in preparation an ointment and was tested

on rabbits. The prepared ointment did not show erythema and oedema formation on the

skin of the rabbits.

 39

Conclusion

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 zone of inhibition 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.

Recommendations

For the further improvement of the presented results of this study, the researchers

thereby recommend the following: (1) improvement of the appearance of the prepared

ointment; (2) test the antibacterial activity of the preparation against Streptococcus

pyogenes and MRSA Staphylococcus aureus; (3) conduct in vitro-release testing for the

prepared antibacterial ointment, (4) conduct stability testing for the prepared antibacterial

ointment and (5) conduct stability test of the excipients and active drug, (6) conduct

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration

(MBC).
 40

REFERENCES
 41

Fawad, et al., (2012) In-Vitro Antibacterial Activity of Piper betle Leaf Extracts.
Journal of Applied Pharmacy. p. 643

Valle, et al., (2016) Antimicrobial Activities of Methanol, Ethanol, and Supercritical CO2
Extracts of Philippine Piper betle L. on Critical Isolates of Gram Positive
and
Gram Negative Bacteria with Transferable Multiple Drug Resistance. p. 1.

Nalina, T., et al., (2007) The Crude Aqueous Extract of Piper betle L. and its
Antibacterial
Effect Towards Streptococcus mutans. pp. 10-11.

Valle, DL., et al., (2015) Antibacterial Activities of Ethanol Extracts of Philippine

Medicinal Plants against Multi-Drug Resistant Bacteria. p. 532.
Ventola, C.L., (2015) The Antibioic Resistance Crisis. pp. 277, 278,

282.

Budiman, A., et al., (2018) Antibacterial Activity of Piper betle L. Extract in Cream
Dosage Forms against Staphylococcus aureus and
Propionibacterium
acne. p.

Aryal, S., (2018) Mueller Hinton Agar (MHA) – Composition, Principle, Uses, and
Preparation.

Pawar, S., et al., (2017) Biochemical Profiling of Antifungal Activity of Betel Leaf
(Piper
betle L.) Extract and Its Significance in Traditional Medicine. Journal of
Advanced Research in Biotechnology. p. 1

Kaveti, B., et al., (2011) Antibacterial Activity of Piper betle Leaves. International
Journal
of Pharmacy Teaching and Practices. p. 129

Blas, E., et al., (2016) Antifungal Property of Polyphenolic Compo unds from Ikmo
(Piper
betle fam. Piperaceae) Leaves and Formulation of Cream. pp. 1, 10, 11,
12

Limuaco O.M, et.al., (2012) Laboratory Exercises in Pharmacognosy and Plant

Chemistry.
pp. 23, 25, 26, 27, 28, 29
Dwivedi V, et. Al., (2014) Journal of Pharmacognosy and Phytochemistry
pp. 93, 94, 94
 42

APPENDICES
 43

APPENDIX A

Plant Sample

Ikmo

Family: Piperaceae

Scientific name: Piper betle Linn.

 44

APPENDIX B

Plant Part Used

Leaves of Ikmo (Piper betle Linn.)

Leaf powder of Ikmo (Piper betle Linn.)

 45

APPENDIX C

Certificate of Authentication
 46

APPENDIX D

IACUC PROTOCOL
47
48
49
50
51
52
53
 54

APPENDIX E

Maceration

Filtration

Evaporation
 55

APPENDIX F

Solubility

Water Acetone

Ethanol Ether
 56

APPENDIX G

Phytochemical Screening

Test for Carbohydrates

 57

Test for Proteins

 58

Test for Flavonoids

 59

Test for Tannins

 60

Test for Saponins

 61

Test for Alkaloids

 62

Test for Glycosides

 63

Test for Resins

 64

APPENDIX H

Microbiological Screening

Staphylococcus aureus

Negative Control

Positive Control

20%
 65

40%

60%

80%
 66

Pseudomonas aeruginosa

Negative Control

Positive Control

20%
 67

40%

60%

80%
 68

APPENDIX I

Antibacterial Susceptibility Test of the Formulated Ointment and the Standard

Drug

Staphylococcus aureus

Negative Control

Positive Control

60% Ikmo Leaf Extract Ointment

 69

Pseudomonas aeruginosa

Negative Control

Positive Control

60% Ikmo Leaf Extract Ointment

 70

APPENDIX J

Statistical Treatment
71
72
 73

APPENDIX K

Preparation of Ingredients

Preparation of White Ointment (based on USP-NF)

White wax………………………………. 2.00 grams

White Petrolatum q.s ad………………… 1000 grams

Modification in the Preparation of the White Ointment using the Ethanolic Crude

Extract of Ikmo

Ethanolic Crude Extract of Ikmo ………. 6.00 grams

White wax ……………………………… 0.02 grams

White Petrolatum qs. ad………………... 10.00 grams

 74

APPENDIX L

Dermal Irritation Test

Day 1

Positive

Negative

111

Day 2

Positive
 75

Negative

Day 3

Positive

Negative

Negative
 76

Day 4

Positive

Negative

Day 5

Positive
 77

Negative

Day 6

Positive

Negative
 78

Day 7

Positive

Negative
 79

APPENDIX M

Plagiarism Scan

SOURCE: https://www.plagscan.com/docman

Summary of Plagiarism Scan

Content Percentage

Chapter 1
The Problem and Its 1846 words 9.1 %
Setting

Chapter 2
Review of Related 1720 words 8.5 %
Literature and Studies

Chapter 3
1715 words 8.8 %
Methods and Procedures