CHAPTER I

The Problem and Its Background

Introduction

Citrofortunella microcarpa (Calamansi) is an important citrofortunella,

meaning that it is an intergenetic hybrid between a member of the genus citrus (in

this case probably the mandarin orange) and the kumquat belonging to Fortunella.

It has considerable amount of pure extract stored in the rind. The most common

methods of extraction are the cold-press method, maceration method, and

centrifugal extraction. The calamansi pure extract is an all-natural body odor

eliminator and antipersperant. Also, Calamansi extract improve the beauty and

quality of skin and hair.

A hand antiseptic, hand disinfectant, hand sanitiser or hand sanitizer is a

supplement or alternative to hand washing with soap and water. Many preparations

are available, including gel, foam, and liquid solutions. The active ingredient in

hand sanitizers may be isopropyl alcohol (isopropanol), ethanol, n-propanol, or

povidone-iodine. Inactive ingredients in alcohol rubs typically include a thickening

agent such as polyacrylic acid for alcohol gels, humectants such as glycerin for

liquid rubs, propylene glycol, and essential oils of plants. Alcohol-based hand

sanitizers are more effective at killing microorganisms than soaps and do not dry

out hands as much.

1
 The purpose of making an antibacterial hand sanitizer from calamansi

(citrofortunella microcarpa) pure extract is to create a new substance and to

discover if this works, to help clean the people’s hands and to be germ free.

2
Statement of The Problem

The main purpose of this study is to utilize Calamansi Pure Extract as an

antibacterial hand sanitizer.

Specifically, it aims to answer the following questions:

1. Is there a significant difference between the hand sanitizer in liquid form with

Calamansi Pure Extract, hand sanitizer in gel form with Calamansi Pure Extract

and commercialized hand sanitizer in terms of the following:

1.1 Texture;

1.2 Smell; and

1.3 Appearance?

2. Is there a significant relationship between the concentration of Calamansi Pure

Extract and the measurement of zone inhibition of Escherichia Coli?

3. Is there a significant relationship between the concentration of Calamansi Pure

Extract and the measurement of zone inhibition of Staphylococcus aureus?

Null Hypothesis

1. There is no significant difference between the hand sanitizer with Calamansi

Pure Extract in terms of the following:

1.1 Texture;

1.2 Smell; and

1.3 Appearance?

3
2.There is no significant relationship between the amount of Calamansi Pure

Extract on the hand sanitizer in terms of the following:

2.1 Texture;

2.2 Smell; and

2.3 Appearance?

3. There is no significant relationship between the amount of Pure Extract,

Escherichia Coli and the Staphylococcus Aureus bacteria zone of inhibition.

Significance of The Study

The study will be of significance to the following:

Students: The hand sanitizer will protect the student’s hands from bacteria.

Also, the hand sanitizer will clean the student’s hands and will give a good smell or

scent.

Future Researchers: The future researchers will inspire students to create a

new product and to discover more about the product. Also, they will make the

students more curious and interested in the product.

Community: The hand sanitizer will be used by the Department of Health in

helping the people who’s in need of keeping the people’s hands clean. Also, the

hand sanitizer will be satisfying for them because the hand sanitizer is antibacterial

and has a scent that will make them feel comfortable.

4
Scope and Limitation

This study aims to produce an antibacterial hand sanitizer from calamansi

pure extract. The product will contain humectants (glycerin), emollients,

emulsifiers, antiseptics (isopropyl alcohol) and calamansi pure extract. The study

will take about a month or so, due to various antibacterial tests and experimenting.

The study will take place at the researchers houses and University of Perpetual

Help System – JONELTA Binan Campus.

The researchers will get the calamansi pure extract strictly from the fruit’s

peelings, and not from the fruit’s seeds, leaves or flesh. The researchers will obtain

the calamansi pure extract through means of maceration.

5
Definition of Terms

Antibacterial properties – is what the researchers will test after making the

product.

Calamansi pure extract– to be used for the product.

Calamansi rind – is where the reasearchers will get the calamnsi pure extract.

Hand sanitizer – is what the researchers will make with the calamansi essential

oil.

Maceration – is a method pretains to the preperation of an extract by solvent

extraction.

6
 CHAPTER II

REVIEW OF RELATED LITERATURE

Related Literature

The researchers are creating a more effective hand sanitizer with

ingredients that are usually found locally. Calamansi is an example of this local

ingredient that is needed for hand sanitizer. Calamondin (Citrofortunella

microcarpa or Citrofortunella mitis) is an important citrofortunella, meaning that the

Calamondin is an intergenetic hybrid between a member of the genus citrus (in this

case probably the mandarin orange) and the kumquat belong to Fortunella.

Calamondin (Citrofortunella microcarpa) is a shrub or small tree growing to

3–6 meters (9.8–19.7 ft). The plant is characterized by wing-like appendages on

the leaf stalks and white or purplish flowers. The fruit of the calamondin resembles

a small, round lime, usually 25-35 mm in diameter, but sometimes up to 45 mm.

The center pulp and juice is the orange color of a tangerine with a very thin orange

peel when ripe. Each fruit contains 8 to 12 seeds. (Potonia, 2014)

Calamansi extract is composed of ascorbic acid, phosphorus and calcium –

the extract is derived from a fruit tree that is native to the Philippines. Today,

women in the Philippines (and around the world) use Calamansi extract to improve

the beauty and quality of skin and hair.

7
 The extract may be found in skin lotions, such as body lotions or cold

creams, or added to shampoos and conditioners. Many women in the Philippines

find the extract of Calamansi an important element of staying youthful looking and

growing long, shiny hair that is both strong and glossy.

Soaps containing Calamansi extracts may also feature fruit extracts with

built-in AHA`s, such as Papaya extract. The combination of Calamansi and other

fruit acids are believed to help the skin shed dead skin cells and reveal a smoother,

rosier and less wrinkled surface. Soaps made with Calamansi may be designed for

use on the body, or especially for the facial area. Sometimes, these soaps will be

used alongside toners and moisturizers that also contain Calamansi.

Shampoos, conditioners and hair products containing Calamansi may be

used to clean, condition and groom the hair. These products will use small

percentages of Calamansi extract, alongside larger concentrations of typical

shampoo, hair conditioner and hair product ingredients, such as aloe vera extract

and mild detergents.

Lotions containing Calamansi may be applied to the face and body in the

morning and at night. Twice daily application of these ingredients allows for

smoother skin, due to the acidic nature of Calamansi, which helps to overturn dead

skin cells. Sometimes, extra sunscreen with UVA.UVB protection should be used,

to counteract the extra sensitivity of the complexion after exposure to fruit acids,

such as Calamansi.

8
 This Filipino beauty secret is found in the splendid tropical regions of the

Southeast Asian island; Calamansi trees grow in abundance, offering Filipino men

and women easy access to this healthful extract (Hayes, 2011)

The Calamansi Plant in the Philippines is as ubiquitous as the Calamansi

Plant is common. The Calamansi Plant is often used to season dishes, preserve

food, remove stains in clothing, deodorize and whiten the skin, and of course,

make into juice. A prevalent ingredient found in most local commercial products,

The Calamansi Plant is definitive and preferred in daily cleansing supplies.

Ironically, what is not as common and certainly not ubiquitous is the Calamansi

(Citrofortunella microcarpa) Essential Oil. ( DancingAlien, 2015,)

A popular seasoning to many Filipino foods, Calamansi is a familiar sight at

Philippine kitchens and homes, and has evolved to a hundred uses in a household.

The Calamansi can be used as a bleaching agent on laundry, a skin whitener,

deodorizer, follicle and hair growth stimulator and many more. The essential oil

comes from the rind of the Calamansi which are extracted either by centrifuge or

steam distillation.

The scent of Calamansi has been proven to help alleviate depression and

anxiety. The Calamansi is also among the few aromatics that not just masks bad

smell, but completely neutralizes them, making the essential oil a great additive to

cleansing products. (Casa de Lorenzo, 2012)

The fruit is used in local recipes in northern Indonesia, especially around

the North Sulawesi region. Fish are spritzed with the juice prior to cooking to

9
eliminate the "fishy" smell. Kuah asang ("sour soup") is a regional clear fish broth

made with calamondin juice. (elevenseven1998117, 2014)

In Florida, the fruit is used in its fully ripe form with a more mature flavor

profile than the unripe version. Tasters note elements of apricot, tangerine, lemon,

pineapple and guava. The peel is so thin, each fruit must be hand snipped from the

tree to avoid tearing. The entire fruit minus the stems and seeds can be used. The

Fruit is hand processed and pureed or juiced and used in various products such as

Calamondin cake, coulis and jam. The peels can be dehydrated and used as

gourmet flavoring with salt and sugar. The fruit was popular with Floridian home

bakers in cake form from the 1920s to 1950s. Once women joined the workforce,

the labor-intensive nature of processing the fruit caused the fruit’s diminished use.

Now that commercially made products and fruit are available, the fruit is

experiencing a resurgence. (Potonia, 2014)

Floridians who have a Calamondin in the yard often use the juice in a

summer variation of lemonade or limeade, as mentioned above, and, left a bit sour,

lemonade cuts thirst with the distinctive calamondin flavor. Also Calamondin can be

used on fish and seafood, or wherever any other sour citrus would be used.

(elevenseven1998117, 2014)

Some say that the plant is native to China and is one of the most cold hardy

citrus. The fruits’ cultivation has spread throughout Southeast Asia, India, Hawaii,

the West Indies, and Central and North America.

10
 In North America, Citrofortunella microcarpa is grown primarily as

an Ornamental plant in gardens, and in pots and container

gardens on terraces and patios. The plant is especially attractive when the fruits

are present.

The plant is frost sensitive and therefore limited outdoors to frost-free

climates (such as Florida, coastal California, South Texas, and Hawaii in the United

States). Potted plants are brought into a greenhouse, conservatory, or indoors as

a houseplant during the winter periods in regions with cooler climates.

However, in the native homeland in Southeast Asia, the Calamondin is easy

to cultivate. The plant grows well in cool and elevated areas and in sandy soils rich

in organic matter.

Waterlogged areas are not suitable for cultivation because calamansi plants

cannot tolerate too much moisture. Calamondin can be propagated by seeds using

the vegetative parts. To produce big, luscious fruits, applying fertilizer, such

as Ammonium sulfate or urea, around each tree one month after planting is

essential. The trees will start to bear fruit one or two years after planting. Trees

have an average life span of five years.

Florida natives, however, report growing Calamondin trees with life spans in

excess of 20 years in some cases, throughout 1950 to the 70's, and throughout the

70's to 90's. So the actual lifespan is an open issue and may depend on climatic

factors, whether the plant is potted or free grown as a citrus yard tree. (Morton, J,

2009)

11
 The Calamondin bears a small citrus fruit that is used to flavor foods and

drinks. Despite the outer appearance and its aroma, the taste of the fruit itself is

quite sour, although the peel is sweet. Eating a whole fruit has a surprise with the

combination of sweet and sour. Calamondin marmalade can be made in the same

way as orange marmalade. Like the citrus fruits, the calamondin is high in vitamin

C. (Dr. Daniel Jacob, 2014)

The fruit can be frozen whole and used as ice cubes in beverages such as

tea, soft drinks, water, and cocktails. The juice can be used in place of that of the

common Persian lime (also called Bears lime). The juice is extracted by crushing

the whole fruit, and makes a flavorful drink similar to lemonade. A liqueur can be

made from the whole fruits, in combination with vodka and sugar. (Susanna Lyle,

2006)

Calamansi is a smooth and slightly spiny plant, growing to a height of 3 to 5

meters. Leaflets are elliptic to oblong-elliptic, 4 to 8 centimeters long. Petioles are

very narrowly or scarcely winged, about 1 centimeter long. Flowers are axillary,

solitary, rarely in pairs, white, and short-stalked. Fruit is yellow when ripe, nearly

spherical, 2 to 3.5 centimeters diameter, 6- to 7-celled, and thin-skinned. The skin

or peel is green to yellowish green or yellow, loosely adhering to the flesh. The

flesh contains a few light orange seeds. (taboradaangela952, 2014)

The Calamansi (citrofortunella microcarpa) is considered as one of the major

fruit crops in the Philippines, which is indigenous to the country. This plant is

characterized by wing-like appendages on the leaf stalks and white or purplish

12
flowers. The fruit has either a spongy or leathery rind with a juicy pulp that is

divided into sections. Calamansi is said to be a good source of vitamin C that that

fruit has been processed into syrups, juices, concentrates, and purees. The juice is

used as flavorings or as an additive in various food preparations to enhance iron

absorption. The juice can also be a preserve ingredient for sweet pickles or

marmalade. Calamansi is easy to cultivate. The plant grows well in cool and

elevated areas and in sandy soils rich in organic matter. Waterlogged areas are not

suitable for cultivation because calamansi plants cannot tolerate too much

moisture. (Potonia, 2014)

Citrus is a common term and genus of flowering plants in the rue family,

Rutaceae. These plants are large shrubs or small trees, reaching 5-15 m tall, with

spiny shoots and alternately arranged evergreen leaves with an entire margin. The

flowers are solitary or in small corymbs, each flower 2-4 cm diameter, with five

(rarely four) white petals and numerous stamens; they are often very strongly

scented. (21food, 2015)

Citrus fruits are notable for their fragrance. The juice contains a high quality

of citric acid giving them their characteristic sharp flavor. The genius is

commercially important as many species are cultivated for their fruit, which is eaten

fresh pressed for juice, or preserved in marmalades and pickles. (Paul

Muljadi,2014)

13
 Hand sanitizers have a form of alcohol, such as ethyl alcohol, as an active

ingredient. It works as an antiseptic. Other ingredients may include water,

fragrance, and glycerin.

Alcohol-based hand sanitizers are clearly a very useful and important

method to prevent most bacterial and viral infections, with rare exceptions.

(Doheny, 2013)

A typical, store-bought hand sanitizer contains the active ingredients

isopropanol or ethanol; these are both forms of alcohol. These types of alcohol

have the ability to kill many germs on contact, without damaging the skin’s tissue.

Isopropanol and ethanol kill germs by dissolving their constituent proteins, causing

the germs to die.

Hand sanitizers have a number of benefits, such as significant reduction of

bacteria on the hands, increased convenience, quick-acting, can cause less

irritation than soap and may improve the skin’s condition. Using a hand sanitizer

can protect your health and help prevent the spread of bacteria. (edensgarden,

2015)

The invention of hand sanitizer was from Lupe Hernandez of Bakersfield,

California. In 1996, Lupe Hernandez called an invention hotline off of the TV, and

ordered a package for an idea that Lupe had on how Lupe would create system in

hospitals, to where all sinks would have an alcohol solutions, at the bottom of the

sink connected to the pipe, outgoing water reaching the hands with cleaning

solution and a sanitizer gel making the system easier for hospital staff and quicker

14
to get patients. This idea came to mind because Lupe was attending school to

become a RN. (Chelsea, 2014)

Related Studies

The main objective of the study entitled “Study of the Antimicrobial Activity of

Lemon (Citrus lemon L.) Peel Extract” conducted by Maruti J. Dhanavade,

Chidamber B. Jalkute ,Kailash D. Sonawane, Jai S. Ghosh, is extraction,

identification of antimicrobial compounds and demonstration of antimicrobial

activity of lemon (Citrus lemon L.) peel against bacteria. As microorganism are

becoming resistant to present day antibiotics, our study focuses on antimicrobial

activity and future prophylactic potential of the lemon peel. Biologically active

compounds present in the medicinal plants have always been of great interest to

scientists. The peel of citrus fruits is a rich source of flavanones and many

polymethoxylated flavones, which are very rare in other plants. These compounds,

not only play an important physiological and ecological role, but are also of

commercial interest because of their multitude of applications in the food and

pharmaceutical industries. The citrus peel oils show strong antimicrobial activity.

The antimicrobial activity has been checked in terms of MIC by using different

solvents against microorganisms like Pseudomonas aeruginosa NCIM 2036 for

which MIC was 1:20 in presence of methanol, for Salmonella typhimurium NCIM

5021 the observed MIC was 1:20 in presence of acetone. In case of Micrococcus

aureus NCIM 5021 the observed MIC was 1:20 when ethanol was used as solvent.

The compounds like coumarin and tetrazene were identified by GC/MS of lemon

peel extract. (Dhanavade, Jalkute , Sonawane, Ghosh, 2011)

15
 In the study entitled “Antimicrobial Activity Of Aloe Vera Leaf Extract”

conducted by Kedarnath, Dr. Kaveri Mahadevappa Kamble, Vishwanath B

Chimkod, C S Patil, The antimicrobial activity of aloe vera extract was tested

against pathogenic bacteria like Staphylococcus aureus, Klebisella pneumonia and

E.coli and fungi like Aspergillus niger and Candida at a dose of 1:20 mg/ml and

2:40 mg/ml by using cup plate diffusion method. Various solvents such as

petroleum ether, chloroform and methanol were used for extracts. The results

reveal that, methanol and petroleum ether at a dose of 20 mg/ml has showed

significant activity against Klebisella pneumonia and E.coli whereas in fungi,

methanol extract showed significant activity against Aspergillus niger and Candida.

Methanol extract has showed maximum inhibitory activity against E.coli and

Candida. Petroleum ether has showed moderate inhibitory activity against

Klebisella pneumonia and Candida. The zone of inhibition was measured and

compared with standard Gentamycin (1 mg/ml). However, in none of the above

mentioned extracts the inhibition zone was not more than that found in standard

i.e., Gentamycin. (Kedarnath, Dr. Kamble, Chimkod, Patil, 2013)

In a study entitled ”Formulation and Evaluation of Novel Herbal Hand

Sanitizer” conducted by Mithun A. Thombare, Babaso V. Udugade, Tushar P. Hol,

Manoj B. Mulik, Dnyaneshwar A. Pawade, Herbal hand sanitizer was prepared

using leaves extracts of Ocimum sanctum, Eugenia caryophyllus and Cymbopogon

flexuous. The antibiotic sensitivity test of the prepared herbal hand sanitizer

against skin pathogens was checked using disc diffusion method and results were

compared with commercially available synthetic hand sanitizer. Results depicted

16
that the herbal hand sanitizer gives larger inhibition zone than the commercially

available synthetic hand sanitizer against Staphylococcus aureus and

Pseudomonas aeruginosa. The efficacy of herbal hand sanitizer was evaluated

using microorganism suspensions; which revealed that the herbal hand sanitizer is

efficient in reducing higher number of microorganism from the hands as compared

to commercial synthetic hand sanitizer. Thus, owing to higher antimicrobial activity

and efficacy these herbal extracts can be used in the preparation of herbal hand

sanitizers on commercial scale. (Thombare, Udugade, Hol, Mulik, Pawade. 2015)

In a study entitled ”Antimicrobial activity of essential oil and various extracts

of fruits of greater cardamom” conducted by Agnihotri S and Wakode S, Greater

cardamom (Amomum subulatum Roxb. Zingiberaceae) commonly known as "Bari

ilaichi" is a well-known plant used in Ayurvedic and Unani medicine. Cardamom

has been used for the treatment of various diseases and disorders like gastric

ulcer. Therefore antimicrobial activity of petroleum ether, methanol and aqueous

extracts from leaves and roots, essential oil and isolated vasicine from A. vasica

were tested against various microorganisms. Antimicrobial activity was done by

disc diffusion method. The zone of inhibition observed was compared with that of

standard drugs, ciprofloxacin and fluconazole. Minimum inhibitory concentration

was determined against microorganisms used in the study. The results of this

study reveal that methanol extract of fruits of A. subulatum shows remarkable

antimicrobial activity against Escherichia coli whereas in case of other

microorganisms used the extract was found inferior to the standard drug used.

Methanol extract of rind showed good antimicrobial activity against Staphylococcus

17
aureus. The essential oil isolated was found effective against majority of

microorganisms used viz. Bacillus pumilus, Staphylococcus aureus,

Staphylococcus epidermidis, Pseudomonas aeruginosa, Saccharomyces

cerevisiae. (Agnihotri S, Wakode S, 2010)

In a study entitled ”Polyherbal Hand sanitizer - Formulation and Evaluation”

conducted by Fatima Grace, Sowmya , Darsika C.,Arul Jothy, S. Shanmuganathan,

The main aim for the preparation of a poly herbal hand sanitizer is for “hand

hygiene”. The preperation is a vital principle in the prevention, control, and

reduction of any acquired infection. Mainly hand sanitizer can stop the chain of

transmission of micro organisms and other bacteria from hand to different parts of

our body. Hand hygiene is important and one of the most critical steps in food

production, food service as well as in homes and other day care preparations.

Hand sanitizer avoids adverse effects like itching, irritation, dermatitis etc. So,

maintaining hand hygiene as the prime criteria-instead of some synthetic

formulation, an attempt has been made to formulate an herbal hand wash by using

some extracts of commonly available plants like And rographis paniculata, ginger,

lemon juice. The formulation was evaluated for the physical parameters. The

formulation is sure that these ingredients on combination behave as an effective

hand sanitizer. (Grace, Sowmya , Darsika., Jothy,. Shanmuganathan, 2015)

In a study entitled ”Formulation And Evaluation Of Herbal Sanitizer”

conducted by Nandkishor S. Wani, Ashish K. Bhalerao, Vikram P. Ranaware, Rahul

Zanje, Hand hygiene is a vital principle and exercise in the prevention, control, and

reduction of healthcare- acquired infections. Right hand washing and drying

18
methods stop the chain of transmission of deadly pathogens(from the

contaminated surface/site) form hands to other parts of the body. Hand sanitization

is the preeminent aid in preventing nosocomial infections caused by different

opportunistic microorganisms and to get this, the use of hand sanitizer becomes

must in recent circumstances. The purpose of present study was to prepare herbal

hand sanitizer incorporating the leaves extracts of Ocimumcanctum Linn.(Tulsi)and

Eucalyptus globulus (Nilgiri), the well-known herbal combination with

multidimensional activities; and to evaluate their respective antimicrobial efficacy

and safety of hands. The formulation was evaluated against the specified

microorganism (Bacteria- E. coli, Pseudomonas aeuroginosa, Staphylococcus

aureus, Bacillus subtilis and Fungi- Sacchromycescerevisiae, Candida albicans) by

culture sensitivity test. The significance was found to be more in comparison to the

standard reference. ( Wani, Bhalerao, Ranaware, Rahul Zanje, 2013)

Justification of the Present Study

This study specifically aims to produce an antibacterial hand sanitizer from

Calamansi pure extract. Calamansi is a type of citrus according to the past studies

which enabled former researchers to produce hand sanitizers from various citrus

fruits, including calamansi. Utilization of the fruits’ rinds were implemented, while

the researchers will utilize the calamansi pure extract.The current study focuses on

the calamansi pure extract's antibacterial properties in order to utilize the pure

extract as an antibacterial hand sanitizer.

19
 CHAPTER III

METHODOLOGY

Materials

The research will need the following materials in making a hand sanitizer:

Blender jar, petri dishes, rotary evaporator, stirring rod, Mueller Hinton agar,

incubator,sterile plastic bottle,100ml of 80 % ethanol alcohol, cotton wool, electric

stove, weighing scale, sterile cotton applicator, sterile hand gloves, distilled water,

filter paper, for the 1st setup :100 grams of calamansi peelings, for the 2 nd setup :

75 grams of calamnsi peelings, and for the 3 rd setup : 50 grams of calamansi

peelings

Procedure

Calamansi Fruits were obtained from the local market. The fruits were

cleansed with distilled water. The peel of calamansi was sliced and were ground

with the blender to form powder samples. The ethanol extracts were prepared by

soaking 100 grams of the peel in 100 ml of 80% ethanol at room temperature for

24 hours. The extracts were filtered after 24 hours through cotton wool. The

extracts were concentrated using a rotary evaporator with the water bath set at 40

degrees Celsius.

Data Gathering Procedure

Sterile Mueller Hinton agar was taken and seeded with different microbial

culture and plates were prepared. Sterile filter paper discs were loaded with the

obtained extract and was taken care that the sterile disc completely absorbed the

extract.
20
 The plate was incubated overnight at 30 degree Celsius. After incubation,

the zones of inhibition was measured and recorded.

21
 CHAPTER IV

DATA ANALYSIS and INTERPRETATION

This chapter includes the data analysis and interpretation of the gathered

data from the experiment. Data was gathered by means of measurement and

computation.

GROWTH INHIBITION
Effect of Calamansi peel maceration on the growth of the Pathogenic Escherichia
Coli

Escherichia Coli exposed in Calamansi Peel Extract (Maceration) in Mueller

Hinton Agar

CONTROL (+) CONTROL (-) DIFFERENT

TRIAL CONCENTRATION
Cefuroxime Distilled Water
50% 75% 100%
1 27mm 0mm 10mm 13mm 15mm
2 26mm 0mm 10mm 12mm 16mm
3 26mm 0mm 11mm 13mm 15mm
MIC 26mm 0mm 10mm 13mm 15mm

Figure 4.1 Measurements of the zones of inhibition of E. coli exposed to

Calamansi Pure Extract (with positive and negative controls)

This table states the different concentrations of the calamansi pure extract

and the measurement of zones of inhibition. The zones of inhibition under 50%,

75%, and 100% concentrations were measured. During the first trial using

Escherichia coli under 50%, at 10 mm were measured, the second trial was at the

same amount, and at the third trial 11 mm were measured. At 75%, using the same

bacteria, during the first trial, 13mm were measured, the second was about 12mm,

and the third was at 13mm. At 100%, during the first trial, 15 mm were measured,

22
the second was 16mm, and the third was at 15mm. The results were compared to

the positive and negative controls namely Cefuroxime for the positive control and

distilled water for the negative control.

GROWTH INHIBITION
Effect of Calamansi peel maceration on the growth of the Pathogenic
Staphylococcus Aureus

Staphylococcus Aureus exposed in Calamansi Peel Extract (Maceration) in

Mueller Hinton Agar

CONTROL (+) CONTROL (-) DIFFERENT

TRIAL CONCENTRATION
Tobramycin Distilled Water
50% 75% 100%
1 20mm 0mm 10mm 11mm 13mm
2 21mm 0mm 11mm 12mm 14mm
3 20mm 0mm 10mm 11mm 14mm
MIC 20mm 0mm 10mm 11mm 14mm

Figure 4.2 Measurement of the zones of inhibition of S. aureus exposed to

Calamansi Pure Extract (with positive and negative controls)

This table states the different concentrations of the calamansi pure extract

and the measurement of zones of inhibition. The zones of inhibition under 50%,

75%, and 100% concentrations were measured. Using staphylococcus aureus

during the first trial, at 50% concentration,10mm were measured, the second at

11mm, and the third was at 10mm. At 75%, the first trial, 11mm were measured,

the second trial at 12mm, and the third at 11mm. Lastly, at 100%, the first trial

measured at 13mm,the second at 14mm, and the third at 14mm. The results were

23
compared to the positive and negative controls namely Tobramycin for the positive

control and distilled water for the negative control.

Measurement
Concentration of
of
Calamansi Pure
Zone of
Extract
Inhibition
X Y x^2 y^2 Xy
50 10 2500 100 500
75 13 5625 169 975
100 15 10000 225 1400
TOTAL: 225 38 18125 494 2975
r= 0.99

Figure 4.3 Computation of Pearson’s Correlation Coefficient, r,

Using Data on the Concentration of Calamansi Extract
and Measurement of Zone of Inhibition (E. Coli)
This table states the gathered data of the correlation analyses in which two

quantitative variables are compared, and to know if there is a significant

relationship or not with the concentration of calamansi pure extract and the

measurement of zone of inhibition for the E. coli. The formula used is r = nƩxy –

(Ʃx)(Ʃy) ÷√ [(nƩx^2-(Ʃx)^2] [nƩy^2-(Ʃy)^2]. The results which is r=0.99 is

compared to the critical value for the correlational analysis which is r=1, when you

get a critical value near r= 1 meaning that there is a significant relationship. And

the alternative hypothesis is accepted.

Measurement
Concentration of
of
Calamansi Pure
Zone of
Extract
Inhibition
X Y x^2 y^2 Xy
50 10 2500 100 500

24
 75 11 5625 121 825
100 14 10000 196 1400
TOTAL: 225 35 18125 417 2725
r= 0.96

Figure 4.4 Computation of Pearson’s Correlation Coefficient, r,

Using Data on the Concentration of Calamansi Extract
and Measurement of Zone of Inhibition (S. aureus)

This table states the gathered data of correlation analyses in which two

quantitative variables are compared, and to know if there is a significant

relationship or not with the concentration of calamansi pure extract and the

measurement of zone of inhibition for the S. aureus. The formula used is r = nƩxy

– (Ʃx)(Ʃy) ÷√ [(nƩx^2-(Ʃx)^2] [nƩy^2-(Ʃy)^2].The results which is r=0.96 is

compared to the critical value for the correlational analysis which is r=1, when you

get a critical value near r= 1 meaning that there is a significant relationship and the

alternative hypothesis is accepted.

25
 Texture Table 1.1
Respondent Set-up Set-up Set-up
1 2 3
1 5 5 3
2 4 5 5
3 5 5 4
4 5 5 3
5 5 5 5
6 5 4 4
7 5 5 3
8 5 4 5
9 5 5 3
10 5 5 3
Total: 49 48 38
Mean: 4.9 4.8 3.8
Grand Average: 4.3667
SSC: 7.4
SSE: 10.1
MSC: 3.7
MSE: 0.3741
F= 9.8904
Figure 4.5 Computed Anova for Texture

This table states the gathered data of the three set-ups for the texture.

There are ten respondents who surveyed for each set-up. The three tables 1.1,

1.2, 1.3 namely the texture, smell and appearance were gathered by means of

getting the anova of each table. The gathered data was analyzed and was

therefore concluded that there is a significant difference between the hand

sanitizer in liquid form with Calamansi Pure Extract, hand sanitizer in gel form with

Calamansi Pure Extract and commercialized hand sanitizer. Since the f ratio value

26
is greater than the critical value which is 3.35 then the the alternative hypothesis is

accpeted while the null hypothesis is rejected or vice versa and that makes up the

decision rule.

Smell Table 1.2

Respondent Set-up Set-up Set-up
Figure 4.6 1 2 3 Computed
1 5 5 3
Anova for 2 5 5 4 Smell
3 5 5 4
This table states the
4 5 5 5
gathered data 5 5 5 4 of the three set-
6 5 5 4
ups for the 7 5 5 4 smell. There are
8 5 5 5
ten respondents 9 5 4 5 who surveyed
10 5 5 3
for each set-up. The three tables
Total: 50 49 41
1.1, 1.2, 1.3 Mean: 5 4.9 4.1 namely the
Grand Average: 4.666667
texture, smell SSC: 4.8667 and appearance
SSE: 5.8
were gathered MSC: 2.4334 by means of
MSE: 0.2148
getting the anova of each
F= 11.3287
table. The gathered data was analyzed and was therefore concluded that there is a

significant difference between the hand sanitizer in liquid form with Calamansi Pure

Extract, hand sanitizer in gel form with Calamansi Pure Extract and

commercialized hand sanitizer. Since the f ratio value is greater than the critical

value which is 3.35 then the the alternative hypothesis is accpeted while the null

hypothesis is rejected or vice versa and that makes up the decision rule.

Respondent Appearance Table 1.3

27
 Set-up Set-up Set-up
1 2 3
1 5 2 5
2 4 4 5
3 5 5 3
4 5 4 5
5 4 4 5
6 4 4 5
7 3 5 5
8 5 4 5
9 4 4 5
10 5 2 5
Total: 44 38 48
Mean: 4.4 3.8 4.8
Grand Average: 4.333333
SSC: 5.0667
SSE: 17.6
MSC: 2.5334
MSE: 0.6519
F= 3.8862
Figure 4.7 Computed Anova for Appearance

This table states the gathered data of the three set-ups for the appearance.

There are ten respondents who surveyed for each set-up. The three tables 1.1,

1.2, 1.3 namely the texture, smell and appearance were gathered by means of

getting the anova of each table. The gathered data was analyzed and was

therefore concluded that there is a significant difference between the hand

sanitizer in liquid form with Calamansi Pure Extract, hand sanitizer in gel form with

Calamansi Pure Extract and commercialized hand sanitizer. Since the f ratio value

is greater than the critical value which is 3.35 then the the alternative hypothesis is

accpeted while the null hypothesis is rejected or vice versa and that makes up the

decision rule.

28
 CHAPTER V

SUMMARY, CONCLUSION, RECOMMENDATION

Summary

29
 The study aims to produce an antibacterial hand sanitizer utilizing calamansi

pure extract. The calamansi pure extract was obtained through the means of

maceration. The antibacterial activity was done by the process of inoculation

against two bacteria cultures, namely: staphyloccocus aureus and escherichia coli.

The data was gathered by measuring the zones of inhibition and was compared to

the positive and negative controls. For staphyloccocus aureus, tobramycin was

used and for escherichia coli, cefuroxime was used as the basis of comparison, in

this case both are positive. Distilled water was used as the negative controls for

both bacteria. As for consumers, their satisfaction was measured through surveys,

which proved the product to be agreeable to the community. Overall, the research

was accepted by both the community and scientific means.

Conclusion
This study concludes that calamansi pure extract has been proven effective

against the bacteria staphylococcus aureus and escherichia coli. The zones of

inhibition under 50%, 75%, and 100% concentrations were measured. During the

first trial using Escherichia coli under 50%, at 10 mm were measured, the second

trial was at the same amount, and at the third trial 11 mm were measured. At 75%,

using the same bacteria, during the first trial, 13mm were measured, the second

was about 12mm, and the third was at 13mm. At 100%, during the first trial, 15 mm

were measured, the second was 16mm, and the third was at 15mm. Using

staphylococcus aureus during the first trial, at 50% concentration,10mm were

measured, the second at 11mm, and the third was at 10mm. At 75%, the first trial,

11mm were measured, the second trial at 12mm, and the third at 11mm. Lastly, at

30
100%, the first trial measured at 13mm,the second at 14mm, and the third at

14mm.

Recommendation
Like all other studies, of course, this research requires further study. Since

the study focuses in the antibacterial activity of the calamansi pure extract, it is

highly recommended to study antimicrobial activity, testing the product against not

only bacteria, but to fungi as well and. It is recommended to utilize different

measurements of calamansi It is also recommended to explore other methods. It is

also recommended to utilize other secretions of calamansi. It is highly

recommended to extend the expiration date of the product. Finally, the researchers

recommend to utilize the calamansi pure extract for other useful materials.

BIBLIOGRAPHY

(Casa de Lorenzo, 2013, blog.com)

31
(DancingAlien, vitalpiece.wordpress.com, 2015)

(Casa de Lorenzo,2012 Casa de Lorenzo.com)

(Julienne Mae Potonia, 2014 ,wwwslideshare.net)

(elevenseven117,2014,www.elevenseven117.wordpress.com/2014/11/13/cala
mondin/)

(bf1,2013,http://bf1.biz/tag/use-of-essential-oil)

( Morton,J.2009, https://en.wikipedia.org/wiki/Calamondin)

(Dr. Daniel Jacob, 2014, https://en.wikipedia.org/wiki/Calamondin)

(
Susanna Lyle 2006, https://en.wikipedia.org/wiki/Calamondin).

(taboradaangela952,2014,https://taboradaangela952.files.wordpress.com/2014
/09/research-ii.docx)

(21food,2015,http://www.21food.com/a-citrus-p68)

(Paul Muljadi , 2014, http://docslide.us/documents/citrus-558466290d749.html)

(Kathleen Doheny , 2013, http://www.webmd.com/cold-
andflu/news/20130208/hand-sanitizers-germs )

(chelsea,2014,http://www.scribd.com/doc/235718433/Investigatory-Project-
CITRUS-FRUITS-AS-A-BACTERIAL-HAND-SANITIZER#scribd

( Meg Hayes, 2011, science.blurtit.com)

(edensgarden ,2015,http://www.edensgarden.com/blogs/news/16317112-make-
your-own-peppermint-hand-sanitizer#.VkxqSHOwrqA)

(Maruti J. Dhanavade,Chidamber B. Jalkute ,Kailash D. Sonawane,Jai S.

Ghosh, 2011, http://www.jourlib.org/paper/2930877#.Vs7irvl97IU)

32
 (Mithun A. Thombare, Babaso V. Udugade, Tushar P. Hol, Manoj B. Mulik,
Dnyaneshwar A. Pawade. 2015, http://www.scopemed.org/?jft=36&ft=36-
1424511810)

(Agnihotri S, Wakode S, 2010, http://www.ncbi.nlm.nih.gov/pubmed/21695005)

( Fatima Grace, Sowmya , Darsika C.,Arul Jothy, S. Shanmuganathan, 2015,

file:///C:/Users/user/Videos/Downloads/Article143-144.pdf)

(Nandkishor S. Wani, Ashish K. Bhalerao, Vikram P. Ranaware, Rahul Zanje,

2013, http://sphinxsai.com/2013/janmar/pharmpdf/PT=06(40-43)JM13.pdf)

(Kedarnath, Dr. Kaveri Mahadevappa Kamble, Vishwanath B Chimkod, C S

Patil,2013,https://www.researchgate.net/publication/257948745_ANTIMICROBI
AL_ACTIVITY_OF_ALOE_VERA_LEAF_EXTRACT)

APPENDIX

Materials

33
34
35
Procedure

Calamansi Fruits were obtained from the local market. The fruits were

cleansed with distilled water. The peel of calamansi was sliced and were ground

with the blender to form powder samples. The ethanol extracts were prepared by

soaking 100 grams of the peel in 100 ml of 80% ethanol at room temperature for

24 hours. The extracts were filtered after 24 hours through cotton wool. The

36
extracts were concentrated using a rotary evaporator with the water bath set at 40

degrees Celsius.

Data Gathering Procedure

Sterile Mueller Hinton agar was taken and seeded with different microbial

culture and plates were prepared. Sterile filter paper discs were loaded with the

obtained extract and was taken care that the sterile disc completely absorbed the

extract.

The plate was incubated overnight at 30 degree Celsius. After incubation,

the zones of inhibition was measured and recorded.

Computations

Computation of Pearson’s Correlation Coefficient, r,

Using Data on the Concentration of Calamansi Extract
and Measurement of Zone of Inhibition (E. Coli)
Measurement
Concentration of
of
Calamansi Pure
Zone of
Extract
Inhibition
X Y x^2 y^2 Xy
50 10 2500 100 500
75 13 5625 169 975
100 15 10000 225 1400
TOTAL: 225 38 18125 494 2975
r= 0.99

This table states the gathered data of the correlation analyses in which two

quantitative variables are compared, and to know if there is a significant

relationship or not with the concentration of calamansi pure extract and the

measurement of zone of inhibition for the E. coli. The formula used is r = nƩxy –

37
(Ʃx)(Ʃy) ÷√[(nƩx^2-(Ʃx)^2] [nƩy^2-(Ʃy)^2]. The results which is r=0.99 is compared

to the critical value for the correlational analysis which is r=1, when you get a

critical value near r= 1 meaning that there is a significant relationship. And the

alternative hypothesis is accepted.

Computation of Pearson’s Correlation Coefficient, r,

Using Data on the Concentration of Calamansi Extract
and Measurement of Zone of Inhibition (S. aureus)

Measurement
Concentration of
of
Calamansi Pure
Zone of
Extract
Inhibition
X Y x^2 y^2 Xy
50 10 2500 100 500
75 11 5625 121 825
100 14 10000 196 1400
TOTAL: 225 35 18125 417 2725
r= 0.96

This table states the gathered data of correlation analyses in which two

quantitative variables are compared, and to know if there is a significant

relationship or not with the concentration of calamansi pure extract and the

measurement of zone of inhibition for the S. aureus. The formula used is r = nƩxy

– (Ʃx)(Ʃy) ÷√[(nƩx^2-(Ʃx)^2] [nƩy^2-(Ʃy)^2].The results which is r=0.96 is

compared to the critical value for the correlational analysis which is r=1, when you

get a critical value near r= 1 meaning that there is a significant relationship and the

alternative hypothesis is accepted.

Respondent Texture Table 1.1

38
 Set-up Set-up Set-up
1 2 3
1 5 5 3
2 4 5 5
3 5 5 4
4 5 5 3
5 5 5 5
6 5 4 4
7 5 5 3
8 5 4 5
9 5 5 3
10 5 5 3
Total: 49 48 38
Mean: 4.9 4.8 3.8
Grand Average: 4.3667
SSC: 7.4
SSE: 10.1
MSC: 3.7
MSE: 0.3741
F= 9.8904

Smell Table 1.2

Respondent Set-up Set-up Set-up
1 2 3
1 5 5 3
2 5 5 4
3 5 5 4
Respondent Appearance Table 1.3
4 5 5 5
Set-up Set-up Set-up
5 5 5 4
1 2 3
16 55 25 45
27 45 45 45
38 55 55 53
49 55 44 55
10
5 45 45 35
Total:
6 50
4 49
4 415
Mean:
7 35 4.9
5 4.15
Grand
8 5 4
4.666667 5
Average:
SSC: 4.8667
39
SSE: 5.8
MSC: 2.4334
MSE: 0.2148
F= 11.3287
 9 4 4 5
10 5 2 5
Total: 44 38 48
Mean: 4.4 3.8 4.8
Grand
4.333333
Average:
SSC: 5.0667
SSE: 17.6
MSC: 2.5334
MSE: 0.6519
F= 3.8862

The three tables 1.1, 1.2, 1.3 namely the texture, smell and appearance

were gathered by means of getting the anova of each table. The gathered data

was analyzed and was therefore concluded that there is a significant difference

between the hand sanitizer in liquid form with Calamansi Pure Extract, hand

sanitizer in gel form with Calamansi Pure Extract and commercialized hand

sanitizer. Since the f ratio value is greater than the critical value which is 3.35 then

the the alternative hypothesis is accpeted while the null hypothesis is rejected or

vice versa and that makes up the decision rule.

40
 RESEARCHER’S PROFILE
Name: Almazora, Thomas Jensen C.
Age: 12
Birthdate: November 16, 2003
Gender: Male
Email Address: jensenalmazora@gmail.com
Educational Attainment: Elementary
Elementary School: Santa Catalina College, Binan Laguna
Awards and Honors Received: 1 st Honorable Mention, Silver Loyalty Award, 3 rd
HEKASI Quiz Bee (group), 1st Math Quiz Bee (activity)
Secondary: Santa Rosa Science and Technology High School

41
 RESEARCHER’S PROFILE
Name: Brigado, Angeliq Melisse T.
Age: 13
Birthdate: December 3, 2002
Gender: Female
Email Address: theempireofsealand@gmail.com
Educational Attainment: Elementary
Elementary School: La Consolacion College – Manila-Binan Campus & University
of Perpetual Help System – JONELTA Binan Campus
Awards and Honors Received: First Honorable Mention, Interschool Spelling Bee
Champion 2014, CEM-English Topnotcher, Excellence Award in Science,
Excellence Award in Music, Most Dilligent, Quiz Bee Champion – English, 5 th Place
in Sci-Math Spelling Bee
Secondary: Santa Rosa Science and Technology High School

42
 RESEARCHER’S PROFILE
Name: Castillo, Rhystal Mei F.
Age: 13
Birthdate: July, 6 2002
Gender: Female
Email Address: rhystal_castillo@yahoo.com
Educational Attainment: Elementary
Elementary School: Emmanuel Christian School
Awards and Honors Received: Best in Arts, Loyalty Award
Secondary: Santa Rosa Science and Technology High School

43
 RESEARCHER’S PROFILE
Name: Garcia, Ken David A.
Age: 13
Birthdate: March 3, 2002
Gender: Male
Email Address: garciakendavid@gmail.com
Educational Attainment: Elementary
Elementary School: Acts Learning Center
Awards and Honors Received: 3rd honorable mention, loyalty award
Secondary: Santa Rosa Science and Technology High School

44
 RESEARCHER’S PROFILE
Name: Haynes, Elijah Carl Camillo A.
Age: 13
Birthdate: August. 21, 2002
Gender: Male
Email Address: senyahelijah21@yahoo.com
Educational Attainment: Elementary
Elementary School: Special Science Elementary Central 3
Awards and Honors Received: Boy Scout Of The Year, Mathscore Award
Secondary: Santa Rosa Science and Technology High School

45
 RESEARCHER’S PROFILE

Name: Madrid, Ckeziah Anne E.

Age: 12
Birthdate: June 3, 2003
Gender: Female
Email Address: kiaanneescobin@gmail.com
Educational Attainment: Elementary
Elementary School: Canossa School & SRES Central 2
Awards and Honors Received: Outstanding Pupil
Secondary: Santa Rosa Science and Technology High School

46
 RESEARCHER’S PROFILE

Name: Ocampo, Monica B.

Age: 13
Birthdate: July 6, 2002
Gender: Female
Email Address: monicabaonocampo@gmail.com
Educational Attainment: Elementary
Elementary School: Saints Peter and Paul Early Childhood Center
Awards and Honors Received: Religious award, loyalty award, drama award, best
in MAPEH, mathscore award, leadership award, civic award, sports award, GSP
ward and consistent honor student.
Secondary: Santa Rosa Science and Technology High School

47
 RESEARCHER’S PROFILE
Name: Reynoso, Czearojohn Drian P.
Age: 13
Birthdate: January 28, 2003
Gender: Male
Email Address: rczearojohndrian@yahoo.com
Educational Attainment: Elementary
Elementary School: Macabling Elementary school
Awards and Honors Received: Boy Scout of the year, 10 th honorable mention, 5th
honor grade 5
Secondary: Santa Rosa Science and Technology High School

48
 RESEARCHER’S PROFILE
Name: Rosales, Liza Marie T.
Age: 12
Birthdate: October 06, 2003
Gender: Female
Email Address: lmrosales@gmail.com
Educational Attainment: Elementary
Elementary School: Holy Rosary College
Awards and Honors Received: Best in Visual Arts, Achiever
Secondary: Santa Rosa Science and Technology High School

49
 RESEARCHER’S PROFILE
Name: Sugita, Yamato C.
Age: 13
Birthdate: October 11, 2002
Gender: Male
Email Address: yamatosugita011@gmail.com
Educational Attainment: Elementary
Elementary School: Santa Rosa Elementary School Central 1
Awards and Honors Received: Huwarang Mag-Aaral and Mathscore Award
Secondary: Santa Rosa Science and Technology High School

50