RELATIONSHIP BETWEEN ANTI-ANGIOGENIC AND ANTI-

INFLAMMATORY PROPERTIES OF SQUID INK POLYSACCHARIDES

(SIPs) FROM Photololigo chinensis IN CHORIOALLANTOIC MEMBRANE

(CAM) ASSAY

A Thesis Submitted

to The Faculty of Pharmacy

University of Santo Tomas

In Partial Fulfillment

Of The Requirements of The Degree

of Bachelor of Science in Pharmacy

by

CLARISSE ANNE S. CAGANDA

MARIE CARMINA T. ERA

MARIA NOERI B. FABELLA

ANGELA C. FERNANDO

PAULA NICOLE G. OPAO

CHRISTIAN RAY C. VILLACRUZES

JOVENCIO G. APOSTOL

NOVEMBER 2014

ACKNOWLEDGMENT

We wish to acknowledge the contribution of the UST- RCNAS in the conduct and output

of this scientific undertaking. We are grateful to the following people for their recommendations

to improve our work: Our Adviser - Prof. Jovencio G. Apostol, Our Proposal Review Panel -

Prof. Ma. Ascuncion Crispina S. Cobar, Prof. Christine Joy Acosta and Prof. Michelle D.

Bartolome.

We wish to thank the technical expertise and support of Prof. Charliemaign Stanley C.

Cruz, ________________ and Prof. Clairefrancis De Guzman.

We dedicate this work to our family and friends for the endless support and

encouragement.

ABSTRACT

Squid ink polysaccharides (SIPs) contain active components that inhibit angiogenesis.

Angiogenesis plays a critical role in normal physiological processes that include inflammation.

In this study, ink from Photololigo chinensis was extracted of its polysaccharide. The structure

from the SIPs obtained from FT-IR and GC/MS consisted of six components, the alpha-L-

sorbopyranose, valeraldehyde, piperidine carboxamide, propanamine, tecasanoic acid and

undecyl ester. The anti-angiogenic and anti-inflammatory property of the SIPs from P. chinensis

was investigated by Chorioallantoic Membrane (CAM) Assay. The CAM Assay results were

quantified using the AngioQuant® software. The SIPs reduced the average vessel length and

branching points to 61.79% and 52.88%, respectively. The anti-inflammatory activity of SIPs

showed a remarkable effect of 77.96% based on the tubular size.

Key words: Photololigo chinensis, Squid Ink Polysaccharide, anti-angiogenic, anti-

inflammatory

TABLE OF CONTENTS

PAGE

TITLE

Oral Defense Endorsement

Certificate of Originality

English Editing Certification

Acknowledgement

Abstract

Table of Contents

List of Tables

List of Figures

List of Appendices

1.0 INTRODUCTION

1.1 Statement of the Problem

1.2 Significance of the Study

1.3 Scope and Limitation

2.0 REVIEW OF THE RELATED LITERATURE

2.1 General Characteristics of Squid

2.2 General Information about Photololigo chinensis (Mitre squid)

2.3 Ink of Sepia esculenta (Golden Cuttlefish) as anti-cancer

2.4 Squid Ink Polysaccharides (SIPs)

2.4.1 Components of SIPs

2.6 Introduction to Angiogenesis

2.6.1 Angiogenesis in Tumor Growth and Cancer Development

2.6.2 Mechanism of the Stimulation and Inhibition of Angiogenesis

2.6.3 Vascular Endothelial Growth Factor (VEGF)

2.7 Introduction to Inflammation

2.7.1 Vascular Endothelial Growth Factor in Inflammation

2.7.2 Mechanism of the Stimulation and Inhibition of Inflammatory Angiogenesis

2.8 The Association of Angiogenesis in Inflammation

3.0 METHODOLOGY

3.1 Study Design

3.2 Methods and Materials

3.2.1 Sample Base and Instrumentation

3.2.2 Collection of Test Animal

3.2.3 Collection of Squid (Photololigo chinensis)

3.3.4 Preparation of Melanin-free SIPs

3.3.5 Chorioallantoic Membrane (CAM) Assay

3.3 Analysis

3.3.1 Instrumental Analysis

3.3.1.1 FTIR Analysis

3.3.1.2 GC-MS Analysis

3.3.2 Data Interpretation and Calculations

4.0 RESULTS AND DISCUSSION

4.1 Characterisation of SIPs

4.2 Anti-angiogenic and Anti-inflammatory activity of SIPs

4.3 Statistical Results

4.4 Discussion

5.0 SUMMARY, CONCLUSION, RECOMMENDATIONS

BIBLIOGRAPHY

APPENDICES

CURRICULUM VITAE

LIST OF TABLES

Table Page

1. Taxonomical Classification of Photololigo chinensis

2. Grouping of Specimen

3. Inflammation Inhibition Scoring

LIST OF FIGURES

Figure Page

1. Photololigo duvauceli Illustration

2. Fourier Transform Infrared Spectroscopy

3. Percent Reduction Based on Mean Junction

4. Percentage Reduction Based on Mean Length

5. Percentage Inhibition of Inflammation

6. Percentage Inhibition Based on Mean Length of Control, Positive Control, Negative

Control and Experimental Groups

7. Percentage Inhibition Based on Mean Junction of Control, Positive Control, Negative

Control and Experimental Groups

LIST OF APPENDICES

A Gantt Chart

B Financial Report

C

D FTIR

E GC-MS

E Raw Data on ….

CHAPTER 1

INTRODUCTION

Background of the Study

Angiogenesis plays a critical role in many normal physiological processes such as cancer

progression in tumor growth, inflammatory diseases, and some systemic autoimmune diseases.

It is an important component in new tissue development, growth and repair. Several studies give

evidence that angiogenesis and inflammation are co-dependent. In these studies, angiogenesis

contributes to the inflammatory pathology through proliferation, migration, and recruitment of

tissue and inflammatory cells which can be damaging to normal tissues. Several angiogenic

mediators, including growth factors, cytokines, cell adhesion receptors, chemokines and

chemokine receptors, have been implicated in the process of capillary formation. There is a

regulatory network in inflamed tissues that is involved in the homeostasis of angiogenesis.

There are studies supporting that squid ink polysaccharides (SIPs) contain active

components that inhibit angiogenesis in tumor metastasis. The inhibition is through the sulfation

with the pyridine-sulfur-trioxide complex in dimethyl sulfoxide. The sulfation of the

glycosaminoglycan (GAG)-like polysaccharides in the squid ink was recognized to possess an

angiotensin-converting enzyme inhibitor.

Statement of the Problem

This study seeks to determine the angiogenesis inhibiting property of SIPs in angiogenesis and

inflammation in Chorioallantoic Membrane (CAM) Assay. Moreover, this paper aims to answer

the following questions:

1. What is the percentage inhibition of angiogenesis provided by SIPs?

2. Will there be a significant difference in the blood vessel growth with the addition of the

following:

a. retinoic acid

b. SIPs

2. Will there be a significant difference in the blood vessel inflammation with the addition

of the following:

a. Hydrocortisone

b. SIPs

3. Is there a correlation between the angiogenesis and inflammation effect of the SIPs?

Conceptual Framework

The process for the entire experimentation is described by the input, throughput and the

output. The input is the collection of materials to be used such as the P. chinensis samples, the

reagents to be incorporated with the SIPs , and the duck eggs to serve as the test subjects. The

throughput is composed of the CAM Assay. Lastly, the output is the gathering of data following

the inhibition of angiogenesis and inflammation by SIPs, the comparison of the control subjects

and the statistical representation of all the results from the experimentation.

Objectives of the Study

● To establish inhibiting properties of SIPs from Photololigo chinensis through

Chorioallantoic Membrane (CAM) Assay

● To correlate the activity of angiogenesis and inflammation

Significance of the Study

    This study aims to contribute to the dynamically evolving branches of science especially in the

field of Pharmacy, and provide information regarding the inhibiting property of SIPs in

angiogenesis and inflammation. In addition, this study is deemed significant to the following:

   

Scope and Limitations

The study will limit its parameters to the structural contribution and inhibiting properties in

angiogenesis in the pathological pathway in mediating inflammation. SIPs used in the study will

be compared to existing pharmacologic agents used to treat angiogenesis and inflammation. This

study does not deal with the mechanism of SIPs in the biologic activity and will not seek to

identify the other effects of SIPs upon administration.

Definition of Terms

Angiogenesis - the formation and differentiation of blood vessels

Inflammation - a local response to cellular injury that is marked by capillary dilatation, leukocytic infiltration, redness, heat, and pain and that serves as a mechanism initiating the elimination of noxious agents and of damaged tissue

Chorioallantoic Membrane (CAM) Assay - in vivo angiogenesis assay

Polysaccharide - a carbohydrate that can be decomposed by hydrolysis into two or more molecules of monosaccharides; especially : one (as cellulose, starch, or glycogen) containing many monosaccharide units and marked by complexity

CHAPTER 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter focuses on studies related to the current research regarding the use of Squid

Ink Polysaccharides (SIPs) in angiogenesis. This review begins with the scientific information

about squids, established uses for SIPs, related source of SIPs, definition and cause of

angiogenesis, factors involving angiogenesis, followed by background of the methodology in the

current research.

General Characteristics of Squid

Squid is a cephalopod which belongs to the order Teuthoidea. Squids are fast swimmers

and are usually found in coastal or oceanic waters. The total length of a squid, including the

tentacles, ranges from 1.5 centimeters to more than 20 meters. They usually comprise of 10 arms

or tentacles which have four rows of suckers with toothed, horny rings (Encyclopedia Britannica,

2014).

According to the Columbia Encyclopedia 6th Edition (2013), the squid is carnivorous and

is one of the most highly developed invertebrates. Its shell is shaped like a quill pen and is the

chief swimming organ of the animal. Two of the 10 arms of the squid used to steer in swimming

are used to hunt its prey. It breathes through its gills and also emits an inky material from its ink

sac, usually when in danger.  A photo of the squid is shown in Figure 1.

Figure 1. Photololigo duvauceli Illustration

General Information about Photololigo chinensis (Indian Squid)

    According to a study by Bat, Vinh, Folkvord, Johannessen, Tsuchiya, and Segawa (2009),

Photololigo chinensis is a neritic species. The species is distributed in the Western Pacific,

around South and East China Sea to Japanese waters, Gulf of Thailand, Arafura, Timor Sea and

northern Australia. They are considered very important among the fishing community because

they occupy 40-50% of the total catch of squid species in the area.  

P. chinensis have arm sucker rings  with about ten sharp, conical teeth on distal margin.

They also have 30 tentacular sucker rings. The taxonomical classification is found in Table 1

below.

Table 1. Taxonomical Classification of Photololigo chinensis

Kingdom Animalia

Phylum Mollusca

Class Cephalopoda

Subclass Coleoidea

Order Theuthida

Suborder Myopsida

Family Loliginidae

Genus Photololigo

Species chinensis

Ink of Sepia esculenta (Golden Cuttlefish) as Anticancer

    Sepia esculenta and Loligo duvauceli have the same class (Cephalopod) and studies about S.

esculenta can serve as source of literature in this study.

    As stated in the study of Ding, Huang, Yang, Yu, and Yang (2011), in traditional Chinese

medicine, S. esculenta ink has been used to treat hemostasis. The ink of S. esculenta showed

antitumor activity against Meth-A fibrosarcoma in BALB/c mice. Different marine mollusks

possess peptides which showed anticancer activity. It was found that the part which had the

anticancer activity was the carbohydrate although such activity was still uncertain.

Squid Ink Polysaccharides

    Polysaccharide is a carbohydrate and is also known as glycan. Its structure is either branched

or linear. It is composed of many molecules of one or more sugars (Encyclopedia Britannica,

2014).

In a study conducted by Chen et al. (2010), squid ink polysaccharides were found to

inhibit angiogenesis in tumor cell metastasis.

The squid ink polysaccharides (SIPs) of Ommastrephes bartrami reported to inhibit

angiogenesis were isolated in vivo using chick embryo chorioallantoic membrane (CAM) assay.

These SIPs are known to possess such effect through sulfation with the pyridine-sulfur-trioxide

complex in dimethyl sulfoxide. The sulfation of glycosaminoglycan (GAG)-like polysaccharides

found in squid ink was recognized to have pharmacological agents and other active components

most importantly, an angiotensin-converting enzyme inhibitor (Chen et al., 2010).

Components

Melanin-derived from amino acids, but is not a protein. A biopolymer that occurs in two

forms: eumelanin and pheomelanin which differ in their molecular precursors. Eumelanin is the

one present in cephalopods ink. It is a polymer of 5,6-dihydroxyindole (DHI) and 5,6-

dihydroxyindole-2-carboxylic acid (DHICA), which are derived from tyrosine and is dark brown

in color.Pheomelanin is composed of the monomers, benzothiazine and benzothiazole, formed

when cysteine is present and with an orange-red color. Some important chemical in melanin

producing pathway in the ink gland: tyrosine,dopamine, and dopamine enzymes.

Peptidoglycan- fucose-rich peptidoglycans have been isolated from ink of several species

of squid. It is derived from enzymatically degraded ink with a resultant molecular fragment-

polysaccharides and oligopeptides.

Amino acids- cephalopod ink is high in dissolved free amino acids. Its potential function

is providing phagomimetic properties to ink which is use as a chemical defense.

Metals- high level of metals, such as cadmium, copper, and lead, have been found in

cephalopod ink. They function as modulators of the activity of enzymes in the melanin synthetic

pathway ( Derby, C., 2014. Cephalopod Ink: Production, Chemistry, Functions and Applications.

Marine Drugs, 12(5), 2700-2730. doi: 10.3390/md12052700 ).

Introduction to Angiogenesis

    Angiogenesis is a normal process wherein new blood vessels form. It happens for the growth

of the body and to replace damaged cells and tissues. Abnormally, it can occur as a factor for

tumor progression. As clusters of cells with no harmful effects, benign tumors may begin to form

new blood vessels, making it turn into a malignant tumor (Encyclopedia Britannica, 2014).

    Angiogenesis involves extracellular matrix degradation, proliferation, survival, migration, and

morphological changes of endothelial cells and their anastomosis to assemble into a vascular

structure (Carmaliet & Jain, 2000).

Angiogenesis in Tumor Growth and Cancer Development

    Angiogenesis is an important factor in tumor growth, invasion, progression, and metastasis as

it helps create blood vessels within and around the tumor by supplying it with blood, oxygen, and

nutrients (Yu et al., 2013).

    Due to the activation of the endothelial cell (EC) proliferation, the lack of oxygen makes the

malignant tumor or cells turn angiogenic and release angiogenic factors and proteolytic enzymes.

This will provide more blood vessels to the tumor, supplying it with enough metabolites to grow.

This complicated network of blood vessels also provides the tumor the opportunity to distantly

metastasize.

Mechanisms of the Stimulation or Inhibition of Angiogenesis

There are various mechanisms which contribute to the stimulation or inhibition of

angiogenesis. According to a study conducted by Kota et al. (2012), among the inducers of

angiogenesis are vascular endothelial cell growth factor signaling pathway, hypoxia, chronic

inflammation, oxidative stress, hyperglycemia, and advanced glycation end products (AGEs),

advanced lipoxidation end products (ALE), and angiotensin.

Angiogenesis occurs due to the broken balance between the net molecules that have

either positive regulators such as the vascular endothelial growth factor (VEGF) or negative

regulators like angiostatin, endostatin, etc. (Zhu et al., 2010). VEGF interacts with its receptors

and each possesses an internal tyrosine kinase that would generate signaling pathways. The

signaling pathways stimulate the endothelial cells’ proliferation and migration. Thus, result to the

induction of angiogenesis (San’t Ana, Gouvea, Nakiae, & Selistre-de-Araujo, 2008).

The inhibition of angiogenesis, as opposed to the mechanisms which stimulate such, are

attributed to factors varying from inadequate bone marrow derived extracellular matrix

(ECM/BM) degradation, growth factor and cytokine imbalance, and signal transduction

problems (Kota et al., 2012). The presence of glycated basic fibroblast growth factor (BFGF) in

diabetic individuals and would cause a great reduction in its ability to activate angiogenesis

(Wang, Chen, Jun, & Hu, 2009). Angiostatin has an anti-angiogenic factor (Zhu et al., 2010).

Vascular Endothelial Growth Factor

    Vascular endothelial growth factor (VEGF) is one of the inducers of angiogenesis. It is a

highly specific mitogen for endothelial cells. Its signal transduction involves binding to tyrosine

kinase receptors and results to endothelial cell proliferation, migration, and new vessel

formation. VEGF encourages microvascular hyperpermeability which precede angiogenesis

(Hobben et al., 2004).

Introduction of Inflammation

Inflammation, as defined in the Merriam-Webster Dictionary (2014), is the body’s local

response to cellular injury that is marked by capillary dilatation, leukocytic infiltration, redness,

heat, and pain and that serves as a mechanism initiating the elimination of noxious agents and of

damaged tissue.

According to the Concise Encyclopedia (2014), it is the local reaction of living tissues to

injury or illness, including burns, pneumonia, leprosy, tuberculosis, and rheumatoid arthritis. Its

major signs are heat, redness, swelling, and pain. The process begins with brief contraction of

nearby arterioles. Dilation follows, flushing the capillaries with blood, from which fluid, plasma

proteins, and leukocytes pass into the injured tissues, causing swelling as they attack the cause of

injury. Initial acute inflammation can have any of four outcomes: resolution, organization,

suppuration, or chronic inflammation. Sometimes treatment—including antibiotics for bacteria,

or surgical removal of an irritating foreign body—can eliminate the cause. If not, anti-

inflammatory drugs (e.g., cortisone or aspirin) may be given, or simple remedies (e.g., hot or

cold compresses) may be applied.

Vascular Endothelial Growth Factor in Inflammation

    VEGF is required in wound and inflammation neovascularization. It is the only well-

characterized secreted mitogenic factor which acts exclusively on endothelial cells. VEGF is

shown in the retina prior to the development of neovascularization, meaning that it is a primary

angiogenic factor.

    PGE1 and PGE2 do not directly stimulate angiogenesis in vitro on endothelial cells but they

are potent inducers of angiogenesis, though the mechanism of action is unknown. PGE2 induces

a rapid induction of VEGF mRNA in synovial fibroblasts.

    Because these agonists are capable of inducing angiogenesis, the induction of VEGF may be

an important mechanism in inflammatory angiogenesis (Ben-Av, Crofford, Wilder, Hla, 1995).

Mechanisms of the Stimulation and Inhibition of Inflammatory Angiogenesis

    Inflammatory and hypoxic fibrinoblasts, monocytes, macrophages and tumor cells are capable

of inducing angiogenic factors such as growth factors and cytokines specifically from

macrophages. Angiogenesis can be induced either in vitro by direct proliferation, migration, and/

or differentiation of endothelial cells or in vivo through indirect intermediary mechanisms

(Jackson et al., 1997).

The Association of Angiogenesis in Chronic Inflammation

Inflammation is the first response of the body's immune system to infection or injury

whereby inflammatory mediators (such as histamine, prostaglandins, and cytokines) are released

to bring about the vascular permeability and cellular changes (Furie, 2014). It attempts to remove

harmful stimuli in order to begin the healing process. Redness (rubor), heat (calor) swelling

(tumor), pain (dolor) and dysfunction of the organs involved (functio laesa) are the primary

results of inflammation.

Chronic inflammation occurs when the body's response failed to eliminate the cause of

acute inflammation. It involves rapid production of new cells and the recruitment of

inflammatory mediators that are highly harmful to normal tissue. In some parts of the

proliferating tissues, hypoxia can induce further capillary development. This is due to the

presence of macrophages which can induce the release of angiogenic factors under hypoxic

conditions. New blood vessels brought about by angiogenesis can sustain the chronic

inflammation by transporting inflammatory mediators and by supplying oxygen of the

proliferating tissues (Feidler & Augustin, 2006).

There is considerable evidence that angiogenesis and chronic inflammation are co-

dependent in health conditions such as rheumatoid arthritis, tumor growth metastasis and

inflammatory bowel diseases. Inhibition of angiogenesis results in anti-inflammatory activity.

Mediators of inflammation can promote angiogenesis, such as interleukins, TNFa,

prostaglandins, and platelet activating factor. Some can change the balance between the

stimulation and inhibition directly or indirectly promoting VEGF as an angiogenic inducer

(Jackson, Seed, Kircher, Willoughby, and Winkler, 1997).

According to Arroyo and Iruela-Arispe in 2010, angiogenesis favors inflammation. It has

a lot of similarities with tumor progression in cancer because it is also a proliferative process.

Proliferation of inflammatory tissue expands due to the increased need for blood supply, to both

provide nutrients and remove waste. Angiogenesis, thus, increases vascular surface area

produces pro-inflammatory mediators and enhances it as well. Inhibition of angiogenesis favors

chronic inflammatory conditions. The presence of inflammatory cells promotes angiogenesis.

Macrophages in inflammatory cells are capable of producing pro-angiogenic molecules,

cytokines and growth factors that is known to regulate angiogenesis. The specific inflammatory

macrophage cell type, MI and M2 are capable of increasing and decreasing angiogenesis,

respectively. These can be found at most sites where angiogenesis is occurring. Thus, inhibition

of inflammatory mediators might as well block the production of pro-angiogenic molecules

Chorioallantoic Membrane (CAM)

Relevance of the Review to the Present Study

The previous researches reviewed in this chapter serves as the backbone of the present

study. The review of the related literature and studies shows the characteristics of P. chinensis or

the Indian Squid and its taxonomical classification. This is useful because it will help the

researchers identify and differentiate this squid from others.

The study of Ding et al., on the other hand, states information about the antitumor and

anticancer activity of S. esculenta, a known relative of P. chinensis. This information can be

greatly used as a basis for finding angiogenesis inhibition properties in the Squid Ink

Polysaccharides of P. chinensis.

The research of Chen et al. is also relevant to the present study because it presents

information about the inhibition properties of Squid Ink Polysaccharides against angiogenesis in

cancer and tumor cells. The studies of Carmaliet and Jain, Yu et al., Zhu et al., Sant’ Ana et al.,

and Kota et al. show vast information about angiogenesis and its action on tumor growth and

metastasis. The previous works of Li et al., Stewart, and Lu et al. exhibit the different factors

involved in angiogenesis such as VEGF. Their studies contribute to the present study. The

definitions from Merriam-Webster Dictionary and Concise Encyclopedia help introduce and

identify inflammation. The study of Fury helps associate inflammation to angiogenesis.

In summary, this review cites information about inflammation which is useful because

the study is about the inhibition of angiogenesis in the said response of the body. Different

mechanisms and factors are also stated above. It presents the use of Sepia ink and Squid Ink

Polysaccharides as anticancer and antitumor agents. Both cancer and diabetes have angiogenesis

as their complication so the related literature regarding such can be used as a basis for this study.

CHAPTER 3

METHODOLOGY

This chapter seeks to present the research strategy, methods, processes and the techniques

applied to the study. This chapter is divided into four main sections.

The first part describes the research design and the collection of animal samples utilized

throughout the study. The following section provides information on the methods applied in the

extraction of SIPs and Chorioallantoic Membrane Assay. Finally, the last section describes the

methods by which results from the study shall be gathered and analyzed.

Study Design

The current research utilized exploratory and experimental methods.

There have only been few studies conducted by previous researchers regarding the

inhibition of angiogenesis and inflammation by the use of SIPs, most of which have focused on

angiogenesis in relation to tumors or cancer cells.

Furthermore, the study applied experimental and quantitative methods as it will employ

collection, summarization, and analysis of data. Figure 1 shows the research study design.

Figure 1. Schematic diagram showing the research design of the experimentation for the

determination of the anti-inflammatory and anti-angiogenic property of Squid ink in duck eggs.

Sample Base and Instrumentation

    The researchers utilized a number of 105 fertilized duck eggs which served as the

experimental subjects throughout the study. The said animal models were divided into seven

groups as presented in Table 2.

Table 2. Grouping of Specimen

Group A Group B Group C Group D Group E Group F Group G

Time control

Solvent Control(0.1%

Dimethyl sulfoxide /

DMSO)

200mcg/mL

Retinoic Acid

(Positive Control) Group

1g/50mLSquid Ink

Polysaccharides

Sodium dodecyl sulfate (SDS)

SDS +Hydrocorti

sone(Positive Control) Group

SDS + SIP

Table 2. Grouping of the different treatments.

The squid (Photololigo chinensis) samples to be used in the conduction of the study shall

be collected in Navotas Fishport and will be submitted to the National Museum of the

Philippines for authentication.

Collection of Test Animal

Philippine mallard (Anas platyrhynchos Linn.) duck eggs were used as the animal models

in the experiment. One hundred and five eggs were purchased at Noel and Unice Balut Producer

in Baliuag, Bulacan The outer surfaces of the eggs were disinfected using 70% ethanol. They

were placed in the incubator at the FLR Laboratory in the University of Santo Tomas. The

incubator was maintained at a temperature of 37°C with a constant humidity. They were

incubated for seven days prior to experimentation and application of the reagents to ensure that

the embryos have already developed enough blood vessels for more visible results. The

immediate environment was cleaned and disinfected before and after use.

Collection of the Squid (Photololigo chinensis)

Nine kilograms of Squid were purchased from Navotas Fishport Complex, Philippines.

They were placed in a thermoplastic container filled ice to preserve the freshness of the sample

to be extracted. A portion of the purchases squids were brought to the National Museum of the

Philippines zoology department for authentication and the rest were brought to the University of

Santo Tomas FLR laboratory for extraction of the ink sack.

Preparation of Melanin-free SIP

Ink sac was separated from the squid and the ink was squeezed out. The squid ink was

diluted with cold deionized water (4oC) (Vate & Benjakul, 2013). Thereafter, it was subjected to

centrifugation at 4,000xg for 30min. Supernatant was referred to as ‘melanin-free squid ink’.

Melanin-free squid ink was digested with 1% papain in Tris-HCl buffer (pH 6.8) at 60oC

for 24h. Digestion was repeated twice. Crude melanin-free SIP was obtained after precipitation

with 95% ethanol (Chen et al., 2010). It was further lyophilized for administration.

Chorioallantoic Membrane Assay

Angiogenesis inhibiting properties of SIPs were determined using Chorioallantoic

membrane (CAM) assay. A total of 105 one-day old duck eggs were utilized and divided into

seven groups. One group served as the time control. Hydrocortisone and Retinoic Acid served as

positive controls for inflammation and angiogenesis inhibition, respectively. DMSO and SDS

served as solvent controls. SDS was used as an inflammatory agent. One group was treated with

SIPs alone and another SDS group was utilized and treated with SIPs after 24 hours (Krenn &

Paper, 2009). These were incubated at 37 °C with a constant humidity. 3-4 ml of albumen was

removed from each duck eggs on the fifth day and incubated for another 3 three days. On the

eighth day of incubation, a 2x2 cm window was created on each egg to expose the CAM.

Sterilized filter paper disks (five millimeter diameter) were saturated with each corresponding

samples (200mcg/ml Retinoic Acid, Hydrocortisone 1g/50mL, 0.1% DMSO, 0.5% SDS, and

1g/50ml SIPs) and one filter paper disk was placed on each CAM (Chen et al., 2010). The

window was sealed using a plastic tape and incubated for another 24 hours for evaluation using

an inflammation inhibition percentage system based on Burgermeister, Paper, Vogl, Linhardt

& Franz (2002) and image analysis using the AngioQuant® software.

Table 3. Inflammation Inhibition Percentage System

% INFLAMMATION INHIBITION DESCRIPTION

<40% No Anti-inflammatory Effect

40% - 55% Uncertain Anti-inflammatory Effect

55% - 70% Weak Anti-inflammatory Effect

70% - 85% Good Anti-inflammatory Effect

>85% Strong Anti-inflammatory Effect

FT-IR Analysis of the Lyophilized SIP

Fourier Transform Infrared (FT-IR) spectroscopy was used to identify the unknown

materials present in the lyophilized squid ink polysaccharide. FT-IR represents a fingerprint of a

sample with absorption peaks which corresponds to the frequencies of vibration between the

bonds of the atoms making up the SIP. Since there are no two compounds produce the exact

same infrared spectrum, FT-IR can provide a qualitative analysis of the kind of material and the

amount present. The FT-IR of the lyophilized squid ink samples were performed by causing

molecular rotation and/or molecular vibrations (stretching or bending of bonds) in the molecules

on a Perkin-Elmer 1600 Series FTIR, which helped to analyze different functional group present

in squid ink. The samples were analyzed from 450 to 4400 cm-1.

About 2 mg of the lyophilized SIP and 200 mg of potassium bromide (KBr) were dried

and ground until the particle size were unified. The mixture was then squeezed to form

transparent pellets. A background spectrum was obtained to identify the traces of gases and the

solvent without the sample. After this, the single-beam spectrum of the SIP was obtained which

is composed of the absorption bands of the SIP as well as the background (solvent). The

spectrum of the SIP was the ratio between the single-beam SIP spectrum and the background

spectrum. The data analysis was done by determining the absorption frequency bands in the

sample spectrum.

GC-MS Analysis

Gas chromatography (GC) is a technique used to separate components of a mixture and

Mass spectroscopy (MS) is used to identify the compounds eluted by GC at molecular level. GC-

MS of lyophilized squid ink samples were heated to separate into individual components and

carried through a column with an inert gas (helium). As the squid ink substances emerge to the

column opening, the mass spectrometer identifies the compound by the mass of the molecule.

Statistical Analysis

Statistical differences between the groups are analyzed using the Student’s t-test.

CHAPTER 4

RESULTS AND DISCUSSIONS

Results

The squid ink extract weighed 103.327g and yielded 7.412g lyophilized squid ink

polysaccharide. A percentage yield of 7.18% was obtained after the lyophilisation.

Characterization of SIPs

Figure 2. Fourier Transform Infrared Spectrum of (a) Lyophilzed Squid Ink Polysaccharide and (b)

Lyophilized Squid Ink Polysaccharide with DMSO

In the FT-IR spectrum of two samples, SIPs and DMSO+ SIPs exhibited the characteristic alcohol O-

H absorption of polysaccharides at 3408.75 cm-1

and 3415.62 cm-1,

respectively.

The signals of SIPs in

the regions of 1670-1600 cm-1

belonging to O-H stretching vibrations were broader than those of SDS +

SIPs. The absorbance band of 1059.75 cm-1

describes C-O alkyl aryl ether stretching vibration while

DMSO + SIPs with 1056.74 cm-1

has an amine group, C-N. The characteristic -C≡C-H: alkynes and C-H

alkanes disappeared in DMSO +SIPs. The monosaccharide component of the polysaccharide as

determined in comparison with the Nototodarus gouldi in the study of Falshaw et al. in 2000. The results

indicated that the principal component of SIPs _____ with a _______ as a side chain. The composition of

SIPs was found to be ________. Finally the analysis with the use of GC/MS revealed the presence of six

components with a molar ratio of

The FT-IR showed that there are O-H, aryl, alkyl, and ether functional groups found in

the squid ink polysaccharide which also confirmed the results obtained from GC-MS.

Tetracosanoic acid and α-L-sorbopyranose showed the presence of O-H group, while the ether

group was shown in 3-methyl-2-butenoic acid, undecyl ester.

Valeradehyde is naturally occurring, found in essential oils and plant distillates. It is use as a

chemical building block in the production of amyl alcohol (n-pentanol) and valeric acid. Minor uses

include resin chemistry, rubber accelerator additive, and fragrance additive ( The Dow Chemical

Company, 2009. Retrieved November 21, 2014 from

http://msdssearch.dow.com/PublishedLiteratureDOWCOM/dh_02b9/0901b803802b9a38.pdf?

filepath=productsafety/pdfs/noreg/233-00597.pdf&fromPage=GetDoc ). Lignoceric acid, also

tetracosanoic acid, is a normal carboxylic acid, a very long chain fatty acid (VLCFA). Very long chain

fatty acids (VLCFAs) are exclusively oxidized in peroxisomes and their levels are significantly increased

in tissues of patients with peroxisomal disorders. It has a biofunction of: Cell signaling, Fuel and energy

storage, and Fuel or energy source. It is a nutrient, stabilizer, surfactant, and emulsifier (HMDB, n.d.

Showing metabocard for Tetracosanic acid. Retrieved November 21, 2014, from

http://www.hmdb.ca/metabolites/hmdb02003 ). N,N,2-trimethyl-2-[(2,2,3-trimethyl-pyrrolidinyl)oxy]-1-

propanamine, this chemical can be used as a solvent in organic synthesis. And it is used as finishing agent

of drugs, paints, pesticides, rubber, fiber, textile and resin. Additionally, it can be used as petroleum

additive and preservative. And it is used in the production of fungicides, fiber and engineering plastics.

Similarly, it can react with N-Phenyl-N'-p-tolyl-4λ4-[1,2,4]dithiazolo[1,5-beta][1,2,4]dithiazole-2,6-

diamine to get 6-Phenylimino-5-propyl-4-p-tolylamino-5,6-dihydro-1H-[1,3,5]triazine-2-thione

( Lookchem, n.d. Propylamine. Retrieved November 21, 2014, from

http://www.lookchem.com/Propylamine/ ). 3-methyl-2-butenoic acid,undecyl ester-unknown use α-L-

Sorbopyranose is a monosaccharide and an eukaryotic metabolite produced during a metabolic reaction in

plants, the kingdom that include flowering plants, conifers and other gymnosperms ( Chemical Entities of

Biological Interest, n.d. alpha-D-sorbopyranose. Retrieved November 21, 2014, from site

http://www.ebi.ac.uk/chebi/chebiOntology.do?chebiId=CHEBI:48677 ). N-methyl-3-Piperidine

Carboxamide is use as reactant for synthesis of: Survival motor neuron (SMN) protein modulators,

Diaminotriazine hNav1.7 inhibitors, Heteroalicyclic carboxamidines as inhibitors of inducible nitric oxide

synthase, Orally available naphthyridine protein kinase D inhibitors, and Phosphodiesterase 5 inhibitors.

It is also use as reactant for identification of small molecular inhibitors of importin-β mediated nublear

import ( Sigma-Aldrich, n.d. 4-Piperidinecarboxamide. Retrieved November 21, 2014, from

http://www.sigmaaldrich.com/catalog/cproduct/aldrich/i17907?lang=en&region=PH ).

Anti-angiogenic and Anti-inflammatory activity of SIPs

Figure 2 shows the percent reduction of the Control group, Squid Ink Polysaccharides

group and Retinoic Acid group based on the mean junction.

Based on figure 2, the SIPs and Retinoic Acid group showed a 47.11774287% and

95.67146575% reduction based on the mean junction, respectively. Mean junctions are the

average number of branching points from the complexes (main blood vessel). The SIPs showed a

weaker angiogenic inhibition by 48% compared to the Retinoic Acid group, which is the positive

control.

Figure ___ shows the percent reduction of the Control group, Squid Ink Polysaccharides

group and Retinoic Acid group based on the mean length.

Based on figure ___, the SIPs and Retinoic Acid group showed a 38.20597169% and

93.77700259% reduction based on the mean length, respectively. Mean length is the

average of the length of the complexes and junctions. The SIPs showed a weaker angiogenic

inhibition by 55.57% compared to the Retinoic Acid group, which is the positive control.

The percent reduction of the mean junction and length of the SIPs showed angiogenic

inhibition but is weaker than the effect of the Retinoic Acid that has a proven use as in

inhibiting angiogenesis. The results obtained were supported by the findings of *insert

name of references*

Figure 3. Percentage Inhibition Inflammation of Sodium Dodecyl Sulfate (Negative Control),

Hydrocortisone (Positive Control) and Squid Ink Polysaccharides (Experimental Group)

Figure 3 shows the percent inhibition of the Hydrocortisone and SIPs.

Based on figure ___, the Hydrocortisone and SIPs showed a 78.92884273% and

77.96078174% inflammation inhibition, respectively. The percentage was based on the

mean of the sizes of the junctions and complexes of the vessels. The SIPs showed a good

anti-inflammatory effect based on the percentage inflammation inhibition of

Burgermeister, Paper, Vogl, Linhardt & Franz (2002).

Figure 4. Percentage Inhibition based on Mean Length of Sodium Dodecyl Sulfate (Negative

Control), Hydrocortisone (Positive Control) and Squid Ink Polysaccharides (Experimental

Group).

Figure 5. Percentage Inhibition based on Mean Junction of Sodium Dodecyl Sulfate (Negative

Control), Hydrocortisone (Positive Control) and Squid Ink Polysaccharides (Experimental

Group).

Statistical Result

Comparison of Control and DMSO

Comparison of Control and Retinoic Acid

Comparison of Control and SIPs

Comparison of Retinoic Acid and SIPs

Comparison of Control and SDS

Comparison of SDS and Hydrocortisone

Comparison of SDS and SIPs

Comparison of Hydrocortisone and SIPs

One Way ANOVA Analysis of Control, Retinoic Acid and SIPs

One Way ANOVA Analysis of SDS, Hydrocortisone and SIPs

Pearson Correlation Analysis

DISCUSSION

CHAPTER 5

CONCLUSION

Summary

The two objectives of this study were: to establish inhibiting properties of SIPs from

Photololigo chinensis and to correlate the activity of angiogenesis and inflammation.

Chorioallantoic Membrane Assay was used to achieve the first objective. Seven groups

were established, namely Control Group, Dimethyl Sulfoxide Group, Hydrocortisone + Sodium

Dodecyl Sulfate Group, Sodium Dodecyl Group, Sodium Dodecyl Group + Squid Ink

Polysaccharides Group, Retinoic Acid Group and Squid Ink Polysaccharides Group.

Conclusion

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