SAMAR STATE UNIVERSITY

Catbalogan City
 A Thesis/ An Undergraduate Thesis
Presented to
The Faculty of College of Nursing and Health Sciences
SAMAR STATE UNIVERSITY
Catbalogan City

Exoskeleton Shrimp (Penaeus vannamie): Chitin- Chitosan weight reduction in

Albino mice

In Partial Fulfillment
Of Requirements for the Degree
Of Bachelor of Science in Pharmacy

By
Huberit, Mary Queen
Morallos, Ma. Eunice
Ragub, Maevhelle
Verino, Eden
Abstract
 Table of Contents

TITLE PAGE
APPROVAL SHEET
ACKNOWLEDGEMENT
ABSTRACT
TABLE OF CONTENTS
REFERENCES
CHAPTER 1 PAGE
1. PROBLEM AND ITS SETTINGS
1.1 Introduction
1.2 Statement of the Problem
1.3 Conceptual Framework
1.4 Scope of Delimitation
1.5 Significance of the study
1.6 Definitions of Terms
2. REVIEW AND RELATED LITERATURE
2.1 Related Literature
2.2 Related Studies
3. METHODOLOGY
3.1 Research Design
3.2 Research Chemical Reagents Used
3.3 Research Laboratory Apparatus
3.4 Maintenance of the Animals
3.5 Materials and Methods
 Preparation of Chitin and Chitosan
 Production of Chitin from shrimp shell waste
 Production of Chitosan from Chitin
3.6 Research Schedule
3.7
 CHAPTER 1

INTRODUCTION
Overweight and obesity are global problem of the World Health
Organization (WHO) it is estimated that more than 1.9 adults aged 18 years and
older are overweight. Of those over 6.50 million people are obese. Medically a
distinction is made between being overweight and obese, Body Mass Index (BMI
– calculated by dividing body weight (kilograms) by height (meter squared), the
prevalence of overweight (adult BMI of between 25 and 29.9) and obesity (BMI of
30 or over. Today 2.1 billion people nearly 30% of the world’s population is
either obese or overweight. The rise in global obesity rates over the last three
decades has been substantial and widespread, presenting a major public health
epidemic in both the developed and the developing world. “Obesity is an issue
affecting people of all ages and incomes, everywhere,” said Dr. Christopher
Murray, director of IHME and a co-founder of the Global Burden of Disease
(GBD). In developed countries, men had higher rates of overweight and obesity,
while women in developing countries exhibited higher rates. Looking at
individual countries the highest proportion of the world’s obese people (13%)
live in the United States, the peak of obesity rates also moving to younger ages
among children and adolescents, obesity has increased substantially worldwide.
Between 1980 and 2013, the prevalence of overweight or obese children and
adolescents increased by nearly 50% (IHME, 2014)
In ASEAN commissioned by Asia Roundtable on food Innovation for
improved Nutrition (ARoFIIN) the Philippines has the second lowest obesity and
overweight prevalence at 5.1 percent and 23.6 percent. But despite of low rates
obesity has a strong impact in the Philippines due to the large number of obese
persons in the country – 18 million Filipinos are obese and overweight (Tacio,
2017). Obesity directly linked to a number of different Health illness such as:
Type 2 diabetes, cardiovascular disease, cancer, osteoarthritis, gastroesophageal
reflux, gallstone, as well as psychological and psychiatric morbidities (National
Clinical Guidelines Centre, 2014). Obesity and overweight were estimated to
have caused 3.4 million deaths, most of which were from cardiovascular causes.
“In the last three decades, not one country has achieved a success in reducing
obesity rates, and we expect obesity to rise steadily as incomes rise in low- and
middle-income countries particular, unless urgent steps are taken to address this
public health crisis.” In the need of finding a possible solution aside from a
healthy diet and proper exercise.
A hard outer shell found in Caridea or commonly known as shrimp which
is made from chemical shell called chitosan is the second most natural
biodegradable organic polysaccharide derived from chitin (found in arthropod
exoskeletons), that has been partially or fully deacetylated. It is composed of
linear chains of β-linked d-glucosamine residues. (Qiongyu Guo, Jennifer H.
Elisseeff, in Principles of Regenerative Medicine (Second Edition), 2011. Among
the medicinal uses of Chitosan it lowers the blood cholesterol and high blood
pressure level, reduce chronic disease. (Posted In life health care on February 21,
2015 by Mukta Agrawal). In recent studies this could be used to replace plastic
coatings in packaging of foods to seal in freshness ranging from water-resistant
paper, to coatings for ceiling tiles and wallboard.
Statement of the Problem

This study determined to prove for a best natural fat absorber in replace to anti-
obesity synthetic drugs that are available in the pharmaceutical industry.
Specifically the study aims to:

1. Find if there is a significant differences in absorbing fat using Caridea

shell, -demineralization and deproteinization chitosan on a water and oil
mixture.
2. Measure the amount of fat that is being absorbed by means of
administering a high fat diet on mice for about 3 to 4 days and performing a
laboratory test on fecal fat excretion or from the blood sample
quantitative test.

Null Hypothesis
There is no significant effect of chitosan as a fat absorber.

Scope and Delimitation

The purpose of this study is to help lower the rate or number of obese
people since many are unable to maintain the weight loss or because they are
programmed to do so: the desire to eat. By pursuing this study we will make
sure that it will be easier for people with this monogenic form of obesity to
control their weight.

The study will be conducted at Samar State University Main Campus

located at Barangay Guindapunan. Researcher choose to conduct the study in
this school because all the equipment and reagents to be used were already in
position and the said school was selected for knowing the efficiency of the study.
There were four (4) albino mice in the cage which are the subject of the study
provided by the researcher.
This study will use the extract of the exoskeleton of shrimp (chitin) on the degree
of fat storage in mice by add mixing the chitosan to thier food.

The study will be conducted at Samar State University Main Campus located at
Barangay Guindapunan. Researcher choose to conduct the study in this school
because all the equipment and reagents to be used were already in position and
the said school was selected for knowing the efficiency of the study. There were
eight (4) albino mice in the cage which are the subject of the study provided by
the researcher.

Conceptual Framework

The conceptual framework will establish to serve a guide of the study. As show
in figure 1. The first box is the independent variable which are the weight of the
exoskeleton shrimp before extracting the chitin, weight of the mice and these
variables are intended to be accurate inorder to complete and have a successive
result of the study. The intervening variables in the second box are among those
methods to be used on the exoeriment including the materials and methods use
in extracting chitin-chitosan, methods on how to extract chitin-chitosan and the
frequency of dosing. The test will work if the mice is obese. Last, the third box
shows the dependent variable which is the result of the mice after the treatment
of chitin-chitosan by mixing it to the food they eat.
Independent Variable Intervening Variable Dependent Variable

 Weight of the
shrimp  Extraction of  Weight reduction
(PenaeusVanname chitin-chitosan of chitin-chitosan
i) in kg from from exoskeleton
 Weight of the exoskeleton shrimp in
exoskeleton shrimp treatment of
shrimp  Frequency of mice in a high fat
 Weight of the dosing diet
mice

Figure 1: Schematic presentation of the research

SIGNIFICANCE OF THE STUDY

This study would be useful to overweight or obese people, teachers, students,

and future researcher.

Obese people- the outcome of the study would provide necessary information to
an effective treatment to achieve weight reduction in overweight and obese
patients. This would also give importance in the light of a growing obesity
epidemic and the known link between obesity and serious health risks.

Teachers- the result of the study the teachers found referring to chitosan as a
slimming agent: “Chitosan: Fat Magnet!” or has found the product itself and
presents it to the students. While showing how she has found the information
from our study the class is also confronted with information claiming the
usefulness of chitosan for reducing weight.
Students- the findings of the study therefore recommend students about the
importance of monitoring their weight and Body Mass Index from time in order
to prevent deviation from healthy weight.

Community- the result of the study is for the people in Catbalogan City that the
raw material of shrimp has an alternative use aside from disposing.

Future Researcher-the outcome of the study is also beneficial to the future

researchers. This study will be the basis that a new theory in learning will arise.
This study will enhance the researcher an idea to create and search for more
available studies about chitosan that can solve the problem which one way or
another affects the community.
 DEFINITION OF TERMS

 Chitosan – a polysaccharide made of glucosamine, naturally present in the

exoskeleton of crustaceans. It resists digestion in the stomach but degrades in the
colon. It is a fat-binding fiber found in aquatic crustaceans.

 Glucosamine – a crystalline compound which occur widely in connective tissue,

especially as a component of chitin.

 Collagen – it is the most abundant protein in the human body, found in the
bones, muscles, skin and tendons. It is the substance that holds the body
together. It also helps connective tissue to be strong and provide cushioning for
various parts of the body.

 Appendages – a projecting part of invertebrate or other living organism, with a

distinct appearance or function.

 Aquatic crustaceans- means relating to water; living in or near water or taking

place in water; does not include groundwater, as ‘aquatic’ implies an
environment where plants and animals live. May also refer to; aquatic animal,
either vertebrate or invertebrate, which lives in water for most or all of its life.

 Arthropods-is an invertebrate animal having an exoskeleton, a segmented body,

and paired jointed appendages.

Compound – is a substance that results from a combination of two or more

different chemical elements, in such way that the atom of the different elements
are held together by chemical bonds that are difficult to break.

 Crustaceans – any of a large class of mostly aquatic manipulate arthropods that

have a continuous exoskeleton or calcareous and chitinous exoskeleton, a pair of
often much modified appendages on each segment, and two pairs of antenna.
 Exoskeleton - a hard outer layer that covers, supports, and protects the body of
an invertebrates animals.

 Fiber- is a type of carbohydrate that the can’t digest. Though most carbohydrates
are broken down into sugar molecules, and instead it passes through the body
undigested.

 Invertebrates – are animals that neither possess nor develop a vertebral column,
derived from the notochord.
 CHAPTER 3

Methodology

Research design

The researcher used the Experimental method research, because it is systematic

scientific approach to research. In which the researcher manipulates one or more
variables and controls and measures any changes in other variable. How these
subject will be need to yield the required data, how the data will collect and how
the data will be analyze.

Sampling and Sampling Size

The researchers used four (4) albino mice in both sexes weighing kg. The mice
were divide into two (2) groups. These were subjected to randomization using
random sampling. Were each member of the population having an equal chance
of being selected as subject. The process of extraction of chitin and chitosan from
shells of crustaceans involvedeproteinization and demineralization.
(NasiahLadchumananarandasiram. et al) (material sciences and application,
January 2012)

The mice were grouped into two (2), each consisting four (4) randomly
selected albino mice:

Group 1: (Positive Control Orlistat (Xenecal) 24.6 mg/kg) 2 albino mice

Group 2: (Experimental – Chitosan 24.6 mg/kg) 2 albino mice

 Maintenance of the Animal

 Four albino mice male and female weighing 30-35 grams were used. This
is bought from the breeder Jay-arr Porta at Catbalogan City. The
 laboratory white mice used were properly handled base on the animal
care act on handling rodents it is clearly identified on cage indicating
strain ,sex, age , and contact person. Procedures performed on the animal
are clearly indicated. Individual identified using fur dyes, the handler talk
quietly see to it (gentling) to reduce stress. Move hands slowly, then the
mice handled at the base of the tail using fingers and apply a scruff hold
to the loose skin between the ears with the fingers and forefingers while
maintaining a grip on the tail, the skin is not being pulled too tightly as
the mice might choke. Materials being administered to mice is given orally
e.g. in water of feed through a variety of routes. The average daily
consumption of feed and water for an adult 25g mouse is 3-5 grams and 4
ml respectively. The mice were exposed to 12h:12 h light dark cycle. The
cage is constructed from a non-toxic, non-absorbable material like
colorless, transparent cage , height of cage allowed our mice to stand on
their hind legs,stretch up fully and climb on the bars of the cage lid.(Dr.
Anne Fawcett BA (Hons) BSc (Vet)(Hons) BVSc (Hons) CMAVA NSW
Department of the Primary Industries, Animal Welfare Unit, West
Pennant Hills)

Materials and Method

Research Chemical Reagents

1. 40%Sodium Hydroxide (NaOH)
2. 40%Sodium Hydroxide (NaOH)
3. 1.25N Hydrochloric Acid (HCl)
4. 1.25N Hydrochloric Acid (HCl)
5. Distilled Water
 6. 100 grams Sodium Hydroxide pellets
7. 250 grams shrimp shell
Research Laboratory Apparatus Used
1. 1000 ml Beaker
2. Water bath
3. Filter paper
4. Glass stirring rod
5. Weighing scale
6. Funnel
7. Graduated cylinder
8. Latex gloves
9. Laboratory gown and goggles

Preparation of chitin and chitosan

The shrimp shell waste will be collected at Pier Dos public Market in Catbalogan
City washed thoroughly to eliminate the exist meats of the shrimp in the shell.
Then washed and clean again with tap water to remove the excess dirt. After
cleaning it was allowed to drip for 30 minutes. Chitin and chitosan were
produced in two steps. First,production of chitin from shrimp wastes and then
production of chitosan from the prepared chitin.

Production of Chitin from shrimp shell

The fresh shell of shrimp or prawn were washed, weighed and taken into a
beaker. two volumes of 1.25N HCl was added to the beaker. After 3 hours the
acid-mix sample was washed with water. Again 2 volumes of 1.25 N HCl was
added and kept it for overnight. The sample was washed with tap water
anddried for few minutes. In this way, the remaining minerals were eliminated
from the shell. Then it was deproteinized by adding 8% NaOH (w/w) and then
heated in the water bath for 1 hour at 70-75 0 C. To remove last protein portion it
was deproteinized again and again by the same method. After deproteinization,
the sample was again washed with tap water carefully and dried in an oven at 8
hrs. The material was then pulverized as chitin.

Production of Chitosan from Chitin

To produce chitosan, the produced chitin was washed and dried for few minutes.
Then 40%NaOH was added and heated at 60-75 degree Celsius for 4 hours.
NaOH was drained; the product was washed with cool potable water for several
times and dried in the oven for 13 minutes. Finally, the product was pulverized
and packed as chitosan. Chitosan is obtained and further dried at room
temperature and stored in airtight container.(S.Z Islam,M. Khan and A.K.M.
Nowsad Alam (2016).

Shrimp shell

Washed then weigh

Add 2 vol of 1.25N HCl for 3 hours

Add again 2 vol of 1.25 N HCl and keep it overnight

Add 40 % NaOH and heat at 70-75 degree Celsius for 1 hour

Wash with water to remove alkali and dry in the oven for 13minutes

Pulverized

Chitin

Add 40% NaOH at 60-75 degree Celsius for 4 hours,agitate well

Drain NaOH and wash several times with

tap water

Dry for 15 minutes in the

oven

Pulverized

chitosan

Figure 1. Scheme for the production of Chitin- Chitosan from the shrimp shell
Preparation for Positive Control Drug

Olistat ( xenical) is a pharmacological agent promoting weight loss in obese via

inhibiting of gastric and pancreatic lipase, an enzyme that is crucial for the
digestion of the long chain triglycerides, which at a three daily dose of 120mg
reduces fat absorption by 30% and has been proven to be useful in facilitating
both weight loss and weight maintenance. Nevertheless, it is known if Orlistat
has any impact on the clinical outcomes of other diseases and its long term safety
is still to be determined. Natural products and their active principles, as sources
for new drug discovery and treatment of diseases, have attracted attention in
recent years. Herbs and spices are generally considered safe and proved to be
effective against various human ailments. (R.H. Mahmoud, W.A. Elnour, January
2013).

Orlistat (Xenical) 5mg (Tiruttani Kuppireddy Padmaja1, Parim Brahma Naidu1,

Ghali Eswara Naga Hanuma Kumar, Saravanan Ganapathy, Meriga Balaji, 2014)
will be weigh using laboratory weighing scale and incorporated to the mice’s
meal.

Experimental Layout

Group A Group B

Treatment in Chitosan 24.6 mg/kg Treatment in Xenecal 24.6 mg/kg

Mice 1

Mice 2
Research Schedule

Day 1-5 : The researcher will receive 10 albino mice and will be placed in its
respective cages. After placing them in its cages, the researcher’s will give
standard fed to normalized their weight. Mice are generally fed a diet containing
low fiber (5%),protein (20%) and fat (5-10%). Feed maybe pelleted or
powdered.(Jhon Hopkins University, Animal Care and Use Committee).

Day 6-28 :the researchers will again give a standard feed and weigh each mouse
before feeding to check their weight.

Day 29-48: the researcher will start giving the mice their heavy diet consisting of
three varieties of food: feeds, cheese and processed meat to increase their weight
from normal to obese.

The researchers will weigh the mice before feeding the mice to monitor their
weight.

Day 49-51: the researcher will start giving the albino mice treatment group:
Chitosan 24.6 mg/kg and 24.6 mg/kg Orlistat.

The researchers will monitor the weight of the mice everyday for three days after
giving the treatment group.

The four albino mice were purposively divided into two groups, each group
consist of two pairs. All are given a high fat diet foods like cheese ,processed
meat and feeds. All these are chopped in small pieces. The first group where the
controlled set up receiving a 24.6 mg in a reduced dosage form from a synthetic
Orlistat (Xenical 120 mg capsule), while the second set up will received a 24.6 mg
a polysaccharide Chitosan via food incorporation.
Acclimitation

Group A Initial 1st day 2nd day 3rd day 4th day 5th day
Mice Weight (g)
1 20 20 19 20 21 21
2 21 21 18 20 21 22
Group B
Mice
1 22 22 20 21 22 22
2 20 20 19 21 20 21

23 days of Standard feeding using feeds

Group A Mice grams grams

Mice 1 27 29
Mice 2 27 28
Group B Mice
Mice 1 28 29
Mice 2 27 26

48 days of high fat diet: cheese, processed meat, feeds

Group A grams grams

Mice 1 34 35
Mice 2 35 36
Group B
Mice 1 35 35
Mice 2 34 35