The Comparison on Antibacterial Activities of Two Citrus Fruit Extracts.

Chapter I

Introduction

“Health is wealth”, a famous quote that most people know. It explains that health
is one of the most valuable thing in this world. The society nowadays tends to focus
more on health and aesthetics. Lots of teenagers are yearning to have the trending
“clear skin” which can be achieved if the person is healthy. Beauty brands and
companies are seeking to find the perfect ingredient that will be placed in their products
for it to be effective. Similarly, health companies are also exploring to have a developed
medicine that can improve the state of immunity and health by reducing the microbes
that make people ill. There are abundant fruits like Lemon and Calamansi that are
suitable and can be used in the certain fields.
Located close to the equator, Philippines is one of the country that has a warm
climate. Lemon and Calamansi typically grows in warm tropical and subtropical climate
which make it bounteous in San Pablo City. When you heard the word “citrus”, the fruits
that quickly comes to our mind is Lemon and Calamansi. The researchers chose the
two citrus fruits as a medium in making a solution to our antibacterial problems because
these fruits are affordable and easy to access.
The two fruits look quite the same but their usage is often different. Lemon is
more often used as a food supplement, while Calamansi is also fit to be applied on the
skin. They are both good in terms of health. These two citrus fruits have anti-bacterial
properties that can help our body fight and eliminate bacteria. Citrus fruits are rich in
Vitamin C which is an essential vitamin, meaning it cannot be produced by body.
Calamansi is affordable unlike the lemon. Calamansi is can be used to dismiss or expel
body odor.
Background of the Study

In today’s busy world, bacteria, viruses and other microbes are widespread which
is the common cause and reason of diseases. Due to searching for the prevention of
these microbes, the researchers compare Calamansi and Lemon.

x Citrofortunella microcarpa (Calamansi) also known as Orange Calamondin

belongs to the citrus family. It is a small, bushy, evergreen tree or shrub which probably
originates from China or the Philippines but cultivation gradually spread to other warm
growing regions like California. Its pulp has a distinctive flavor and is described as a
cross between lime and orange. Its flesh is juicy and acidic. Calamansi is a rich source
of Vitamin C and certain antioxidants such as Limonene, as well as Potassium, Vitamin
A and Calcium. Although the juice of this fruit is incredibly sour, many people still
consume it on a regular basis due to its many impressive health benefits. The top
benefits of Calamondin juice include its ability to improve the immune system, soothe
the acidity in the stomach, aid in weight loss, stimulate growth and repair, bleach the
skin, detoxify the body, lower cholesterol and manage diabetes, and give relief from
respiratory infections among others.

Citrus x limon (Lemon) are produced on a small evergreen tree that is native to
regions in Asia. It is a popular fruit that people use in a small quantity and with herbs
and spices but they are really consumed alone (due to their intense sour flavor). They
give flavor to many sauces, salad dressings, marinades, drinks, desserts and it is also a
good source of Vitamin C. In 1747, James Lind found that lemons were extremely
effective at treating the disease which was common among sailors that time such as
Scurvy (serious condition that results from a Vitamin C deficiency). Lemon is a powerful
antioxidant which are thought to boost health and well-being in several ways. Vitamin C
is needed to produce collagen, it may help relieve asthma symptoms and it may be a
protection against cancer. Lemon oil has antimicrobial properties, making it excellent at
reducing many types of bacteria, viruses, and fungus. In addition, lemon contains small
amounts of Thiamine, Riboflavin, Vitamin B6, Pantothenic acid, Copper and
Manganese.
Although Lemon and Calamansi have the same look and taste their properties
are still different. Nevertheless, people can still use Calamansi as a substitute for
Lemon in flu protection, common cold, weight loss, removing body odor and maintaining
a healthy mouth.
Review of Related Literature

Study Antimicrobial Activity of Lemon (Citrus Lemon L.) Peel Extract

Even though pharmacological industries have produced a number of new antibiotics in
the last three decades, resistance to these drugs by microorganisms has increased. In
general, bacteria have the genetic ability to transmit and acquire resistance to drugs,
which are utilized as therapeutic agents (Gislene et al., 2000). For a long period of time,
plants have been a valuable source of natural products for maintaining human health.
The use of plant extracts and phytochemicals, both with known antimicrobial properties,
can be of great significance in therapeutic treatments (Seenivasan et al., 2006). Many
plants have been used because of their antimicrobial traits, which are due to
compounds synthesized in the secondary metabolism of the plant. These products are
known by their active substances e.g. the phenolic compounds which are part of the
essential oils, as well as tannin (Tyagi and Malik, 2010). Essential oils are more
effective in controlling biofilm cultures due to their better diffusibility and mode of contact
(Al-Shuneigat et al., 2005). Hence the essential oils and other extracts of plants have
evoked interest as sources of natural products. They have beenbscreened for their
potential uses as alternative remedies for the treatment of many infectious diseases
(Tepe et al., 2004; Dorman and Deans, 2000).
Lemon is an important medicinal plant of the family Rutaceae. It is cultivated mainly for
its alkaloids, which are having anticancer activities and the antibacterial potential in
crude extracts of different parts (viz., leaves, stem, root and flower) of Lemon against
clinically significant bacterial strains has been reported (Kawaii et al., 2000). Citrus
flavonoids have a large spectrum of biological activity including antibacterial, antifungal,
antidiabetic, anticancer and antiviral activities (Burt, 2004; Ortuno et al., 2006).
Flavonoids can function as direct antioxidants and free radical scavengers, and have
the capacity to modulate enzymatic activities and inhibit cell proliferation (Duthie and
Crozier, 2000). In plants, they appear to play a defensive role against invading
pathogens, including bacteria, fungi and viruses (Sohn et al., 2004). Flavonoids are
generally present in glycosylated forms in plants, and the sugar moiety is an important
factor determining their bioavailability. Preparation from peel, flowers and leaves of
bitter orange (Citrus aurantium L.) are popularly used in order to minimize central
nervous system disorders (Pultrini et al., 2006).The peel of Citrus fruits is a rich source
of flavonoid glycosides, coumarins, $ and (- sitosterol, glycosides and volatile oils
(Shahnah et al., 2007). Many polymethoxylated flavones have several important
bioactivities, which are very rare in other plants (Ahmad et al., 2006). In addition the
fiber of citrus fruit also contains bioactive compounds, such as polyphenols, the most
important being vitamin C (or ascorbic acid), and they certainly prevent and cure vitamin
C deficiency-the cause of scurvy (Aronson, 2001). Antimicrobial activity of the peel
extract is directly concerned with the components that they contain. The studies showed
that essential oils, protopine and corydaline alkaloids, lactons, polyacetylene, acyclic
sesquiterpenes, hypericin and pseudohypericin compounds are effective toward various
bacteria. Nevertheless, other active terpenes, as well as alcohols, aldehydes, and
esters, can contribute to the overall antimicrobial effects of the essential oils (Keles et
al., 2001). The lemon peel extracts in different solvents such as ethanol, methanol and
acetone were subjected to antibacterial assay. The extract in solvent ethanol shows
higher antimicrobial activity against tested microorganisms in comparison with the
extracts of lemon peel in other solvents like methanol and acetone. The aim of this
study was to evaluate the potential of plant extracts and phytochemicals on standard
microorganism strains by using routine antibacterial assay techniques.

ANTIBACTERIAL ACTIVITY OF Citrus limonON Acnevulgaris (PIMPLES)

Pharmacological industries have produced a number of antibiotics in the last three
decades; resistance to these drugs by microorganism has increased. In general,
bacteria have the genetic ability to transmit and acquire resistance to drugs which are
utilized as therapeutic agents [11]. For a long period of time, plants have been a
valuable source of natural products for maintaining human health. The use of plant
extracts and photochemical, both with known antimicrobials can be of great significance
in therapeutic treatment [19] many plants have been used because of their antimicrobial
traits which are due to compounds synthesized in the secondary metabolism of the
plant. These products are known by their active substances e.g the phenolic
compounds which are part of the essential oils as well as tannin [22]. Essential oils are
more effective in controlling biofilm cultures due to their better diffusibility and mode of
contact [3] .Hence the essential oils and other extracts of plants have evoked interest as
source of natural products. They have been screened potentiauses as alternative
remedies for the treatment of many infectious diseases [21]; [8]. Citrus limon is an
important plant of the family Rutaceae. Its cultivated mainly for its alkaloids, which are
having anticancer activities and the antibacterial potential in crude extracts of different
parts Viz leaves, stem, root and flower)of lemon against clinically significant bacterial
strains have been reported .Citrus flavonoids have a large spectrum of biological activity
including antibacterial, antifungal, antidiabetic, anticancer and antiviral activities [4].
Flavonoids can function as direct antioxidants and free radical scavengers. They also
have the capacity to modulate enzymatic activities and inhibit cell proliferation [9]. In
plants; they appear to play a defensive role against invading pathogens including
bacteria, Fungi and Viruses [14]. Many polymethoxylated flavones have several
important bioactivities which are very rare in other plants [2].In addition the fiber of citrus
fruit also contains bioactive compounds such as polyphenols, the most important being
Vitamin C,and they certainly prevent and cure Vitamin C deficiency-the cause of scurvy
[6]. Antimicrobial activity of the peel extract of lemon is directly concerned with the
Components that they contain. Acne vulgaris, Pimple, Zit or Spot is a most common
skin disorder of pilosebaceous unit that affects areas containing the largest oil glands
including the face, back and trunk [1]. Propionibacterium acnes have been described as
an obligate anaerobic organism. Its implicated in the development of inflammatory acne
by its capability to activate complements and by its ability to metabolize Sebaceous
triglycerides into fatty acids which chemotactically attract neutrophils [1]. Long term use
of antibiotics against acne is outdated because of exacerbated antibiotic resistance [5].
The development of antibiotic resistance is multifactorial, including the specific nature of
the relationship of bacteria to antibiotics, how the antibacterial is used, host
characteristics and environmental factors. To overcome the problem of antibiotic
resistance, medicinal plants have been extensively studied as alternative treatment for
diseases [5] This work was designed in order to investigate the Antibacterial activity of
Citrus limon on Acne vulgaris (Pimple). The idea came up considering how Acne
vulgaris is becoming a problem among people. Individuals spend a lot of their money in
buying anti-pimple agents (Cleansers, Soaps, and Creams etc), that contains a lot of
Chemicals which may be Detrimental to the skin. In view of this, the research was
intended to Compare and see the effect of the natural agent (Lemon) with a
conventional agent (Cleanser).
From the research, a total of three species of organisms were isolated from the face,
neck and back of individuals having pimples using sterile swab sticks. The organisms
include Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus
aureus. It was found thatPropionibacterium acnes has the highest percentage frequency
of (58.8%) followed by Staphylococcus aureus(23.5%) and Staphylococcus
epidermidis(17.6%). Table 1 indicated the results of microscopy which showed that
there were 10 gram positive rods and 7 gram positive cocci, although in two of the
samples (Sample K and Sample L) there were both gram positive rods and gram
positive cocci. Table 2 showed the result of biochemical analysis conducted. From the
result Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus
aureus were identified. Table 3 indicated the results of frequency of occurrence of
bacteria isolated from the face, chests and back of individuals having pimples. Table 4
showed the percentage of frequency of occurrence of different bacteria isolated from
chest, face and back of individuals having pimples. Table 5 showed the antibacterial
activity of Citrus limon on pimples .The results showed that Citrus limon was effective at
all concentrations (20%, 40%, 60%, 80% and 100%) used on Propionibacterium acnes,
as the concentration of Citrus limon increases the zone of inhibition also increases.
Table 6 indicated the antibacterial activity of Cleanser on pimples. The result indicated
that at concentrations of 20% and 40% there was no zone of inhibition. The zone of
inhibition was observed at higher concentrations of 60%, 80% and 100%. Table 7
showed the Minimum inhibitory concentration (MIC) of Lemon on Propionibacterium
acnes, The results indicated that as the concentration of Lemon increases an absence
of growth was observed due to the broth becoming less turbid. Table 8 also showed the
Minimum inhibitory concentration (MIC) of Cleanser on Propionibacterium acnes. The
minimum bactericidal concentration (MBC) carried out on Propionibacterium acnes
using Lemon and Cleanser showed no growth.
FRUIT PEEL SOAP AND ITS ANTIBACTERIAL PROPERTIES IN SKIN
CAREINTRODUCTION
Fruits are nature’s wonderful gift to mankind. Since time immemorial fruits have been
the source of food for human beings as they used to reap the harvest of fruits and enjoy
their flavour and taste. Fruits are also the chief source of some of the essential vitamins
and minerals. Fruits are one of the largest groups of food that contain antioxidants and
serve as a natural remedy for a multitude of ailments such as soothing itchy skin,
healing wounds, and protecting our skin from sun damage. Fruits and vegetable wastes
and their by-products are formed in great amounts during industrial processing and
hence represent a serious problem, as they exert a harmful effect on the environment.
So they need to be managed or they can be utilized [1]. Apart from consumption, fruits
are used in cosmetics, soaps, shampoos, toothpaste, etc. Soap: The first concrete
evidence we have of soap-like substance is dated around 2800 BC., the first soap
makers were Babylonians, Mesopotamians, Egyptians, as well as the ancient Greeks
and Romans. All of them made soap by mixing fat, oils and salts. Soap wasn't made for
bathing and personal hygiene but was rather produced for cleaning cooking utensils or
goods or was used for medicine purposes. Today, there are different soaps made for a
vast array of purposes and is available for personal, commercial and industrial use.
There are handmade and commercially produced soap, which are used to wash
clothes, dishes, cars etc. Overall, soap is a remarkably useful substance, just as it has
been for thousands of years [2]. Phytochemicals are plant chemicals that have
protective or disease preventive properties. Plants produce these chemicals to protect
themselves but recent research demonstrates that they can also protect humans
against diseases. There are many phytochemicals and each works differently. These
are some possible actions: Antioxidant, Hormonal action, Stimulation of enzymes,
Interference with DNA replication, Anti-bacterial effect and Physical action. The fruit
peel soaps prepared from different fruits were analysed for the antibacterial properties,
using paper disc method against three bacteria Escherichia coli, Staphylococcus aureus
and Staphylococcus epidermidis in two extract water and ethanol and pH studied.

Calamansi Face Care Research

Introduction Many teenagers nowadays often fuss and whines about their face. They
are more conscious when it comes to their appearance. As teenagers, many adolescent
boys and girls suffer from pimples and acne. Seeing the unwanted pimple, they buy
many kinds of beauty and face care treatment which costs a lot of money. Furthermore,
these commercial chemicals are often irritating to the skin and cause even worse effects
to the skin. Seeing this problem, the researcher immediately thought about a skin care
remedy that not only is cheap, but less irritating and most of all, effective. The
researcher was trying to make use of cheap and easy to find ingredients for his ultimate
elixir. As he was trying different concoctions and mixing loads of mixtures, he was most
amazed in the effectivity of Calamansi (Citrofortunella microcarpa) and egg white
mixture. He also found out that not only these kitchen regulars make useful ingredients
for delicious food, but also an effective component in skin care treatments. Now, these
delectable food ingredients will be tested as a breakthrough face treatment components
and might change the face of skin treatment products everywhere. Background of the
Study As the researcher sees that pimples and acne are common face problems, he
immediately thought of a cheap but effective elixir that is safe to use and not skin-
irritating. He discovered the potential of Calamansi and Egg whites. Therefore, the
study, Calamansi (Citrofortunella microcarpa) Fruit and Egg Whites as Face Treatment
was at hand. It was studied that the Calamansi and egg white has skin care properties
and can promote healthy, young skin.
A teaspoon of both ingredients was applied to the face as a mask and was rinsed off.
After a week of repeating the regime, it was seen that the ingredients have potential in
skin care and face treatment. Conclusions After the study was done, it was found out
and concluded that the Alternative Hypothesis, Calamansi fruits and egg whites are
effective for face treatment, is correct and the null hypothesis wrong. Reccomendations
For future improvements of this study, the researcher recommends to compare this
regime to other commercial skin care procedures and products.

The antimicrobial activity of citrus fruit extracts

The scarcity of local research on extracts of citrus fruits precludes their use as an
alternative medication, particularly in the field of Dermatology. This study aims to
determine the antimicrobial activity of citrus fruit extracts and to contribute to existing
data supporting the use oi these extracts against superficial bacterial and fungal
diseases of the skin. Crude ethanolic pulp and/or seed extracts and essential oils of
Citrus paradise (grapefruit), Citrus grandis (pomelo), and Citrofortunella microcarpa
(calamansi) were screened for antimicrobial activity against Staphylococcus aureus,
Pseudomonas aeruginosa, and Candida albicans the disc diffusion method. Minimum
inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C.
microcarpapulp extract against S. aureus was then determined using standard dilution
susceptibility testing. Thin layer chromatography was used to identify the presence of
flavonoids, using naringin as a standard. The widest zones of inhibition against the test
organisms were seen with calamansi pulp extract. Susceptibility testing showed that
calamansi pulp extract was bacteriostatic for S. aureusup to 12.5 percent dilution.
However, no bactericidal effect was observed. Naringin was identified only in the pulp
extract of grapefruit. In vitro, calamansi pulp extract was shown to be an effective
antimicrobial against S. aureus, P. aeruginosa, and C. albicans. Whether or not this has
any dermatologic significance awaits further /w-vj'votesting.
Conceptual Framework

This research study will use Calamansi (x Citrofortunella microcarpa) and Lemon
(Citrus x limon) extracts as the independent variables. The two citrus fruits are
bactericidal which means they can both kill germs and bacteria. The controlled variables
for the experimental setup are used to compare the two citrus fruits if they reduced or
multiplied the microbes. They both have antibacterial properties that can help to reduce
the microbes.

Independent Variable Dependent Variable

Citrus Fruit Extracts

 Lemon (Citrus × limon) Antibacterial Activities
Extracts
 Reduced
 Calamansi
 Multiplied
(× Citrofortunella
microcarpansi)
Extracts

Figure 1. Research Paradigm

Statement of the Problem

Skin is the largest organ in our body, it comprises about 16% of a person's body
weight and covers a surface area of approximately 22 square feet (2 square meters).
With that large amount of covered part in our body, skin is the most prone to bacteria.
We can get bacteria everywhere — at home, interacting with others, staying outside and
also by just doing nothing.
This study aimed to discuss the comparison on the antibacterial properties
between two citrus fruits which is the Citrus x limon (Lemon) and x Citrofortunella
microcarpa (Calamansi). This will lead the researchers to know which is more effective
in killing bacteria between the two citrus fruits.

This study answered the following questions:

1. Which is more effective citrus to reduce bacteria?
2. What is the advantage of Calamansi and lemon in terms of antibacterial?
3. What does citrus fruit contains to reduce bacteria?

Hypothesis of the Study

Calamansi and Lemon became the most efficient antibacterial than other citrus
fruits. There are no difference on their antibacterial activities.

Scope and Limitations

This study is only limited on determining the difference between the two citrus
fruit extracts. There are two citrus fruits that are exposed on this study. First, the x
Citrofortunella microcarpa (Calamansi) which is used as ingredients on condiments.
Second, the Citrus x limon (Lemon) which is more expensive and is commonly used in
business especially on malls. The instrument that will be used in this study is
microscope. This will be the tool on comparing the antibacterial properties of the two
citrus fruit extracts.
 The outcome will be a great alternative way on discovering the one which is more
effective to be used on the skin. The collection of data started on July 2019 to October
2019 while determining the findings. The results are done in St. Therese Montessori
School of San Pablo Inc.

Significance of the Study

This research will provide information about the comparison of two citrus fruit
extracts which are x Citrofortunella microcarpa (Calamansi) and Citrus x limon (Lemon).
This study will be beneficial to the following individuals:

Students- This will serve as their guide and reference on making research about citrus
fruits. There are students that are currently undergoing adolescence and are having a
problematic skin. There are many skin diseases that the citrus fruits can cure and the
use of it is advisable by dermatologists. Through this study, the students will have an
overview about the comparison of the two citrus fruits.

Community- This will serve as an awareness of every individual in the society which
they will find out that the other essential for antibacterial are citrus fruits. It will also be
an eye-opener for children and even parents on how do citrus fruits give cure to the skin
and become helpful to those who are caring for their skin.

Future Researchers- This will serve as a reference point and an additional knowledge
for further studies related or dealing with citrus fruits and antibacterial properties. This
may help researchers on conducting another study to improve the comparison on two
citrus fruit extracts.
Definition of Terms

For the clarification, the important terms that are used in the study are defined.
The following terms are:

Lemon which is scientifically known as Citrus x limon and is commonly

used in business specially in the mall.

Calamansi which is scientifically known as x Citrofortunella microcarpa is

normally used as ingredient on condiments.

Antibacterial the trait of being bactericidal or being bacteria reducing.

Reduced to make something smaller in number.

Multiplied to make something increase greatly.

Citrus Fruits sour fruits that are rich in Vitamin C.