Review of Related Literature

This portion consists of the following parts: I) Cancer, where its definition and

types, severity, prevalence, factors causing it, stages of its progression, common

treatment for cancer and related studies about cancer is presented; II.) Angiogenesis,

where its definition and importance, pathologic state, examples and importance of

antiangiogenic products, methods of screening for angiogenic products and related

studies were presented; III) Anti-Oxidative Property, where its definition and

importance, antioxidative products, methods of screening and studies were presented

IV) Thunnus albacares, where its taxonomic classification, distribution,

morphological structure and related studies were discussed V) Bile, where its

definition and purpose, chemical components, and procedure of extraction were

presented, as well as its connection to angiogenesis, antioxidants and related studies

about it.

Cancer

Definition and types of cancer. Cancer is considered a disease when a cells

grows uncontrollably and invades other cells, sometimes spreading form one parts of

the body into another (metastasis). This is one word is just use to describe over 100

diseases. It should be clarified that only malignant tumors are cancerous and it is not

contagious (National Cancer Institute, 2019; Cooper, 2000). Generally it is divided

unto main types which depends on what tissue it first occurred. When it produces on

skin or epithelial tissues, it is known as carcinomas. When it is produced on

connective and supportive tissues like bones, cartilage and blood vessels, it is known

as sarcoma. When it occurs on blood forming tissues like in red marrows, it is known
as leukemia. When it occurs on nervous system, it is named as lymphoma (Cooper,

2000; Shiel, 2018). This information is important since it is necessary to clearly

define cancer and be aware of its types.

Severity and Prevalence of Cancer. World Cancer Research Fund (2019)

said that worldwide in 2018, over 17 million new cases of cancer was diagnosed with

lung cancer topping the list followed by breast cancer, both amassing around 12.3

million cases. In women, breast cancer lung cancer and colorectal is the most

prevalent while for male, its lung, prostrate and colorectal cancer. Almost the same

pattern were observed in the Philippine setting. As of 2018, 141 021 cases were

diagnosed and the death tolled at 86 337 at that year alone. Most of these cancer

patientsare have breast, lung liver, colorectal and prostrate cancer, with breast topping

the list for women and lung cancer for men. (The Global Cancer Observatory, 2019).

This information holds bearing in this study since this information will convince the

readers that the problem for cancer is indeed widespread and serious .

Factors Causing Cancer. A lot of factors may induce cancer such as

chemical factors (carcinogens), radiation and even viruses. Some of this factors

induce cancers by altering DNA structure, causing mutations. This may be induced by

radiation like prolonged exposure to ultraviolet rays, aflatoxin ( produced by molds

like Aspegillus niger causing liver cancer) and carcinogens from cigarretes which was

said to induce one third of all cancer related deaths (Cooper, 2000; Ames, Swirsky-

Gold, & Willet, 1995). However, cancer will also occur if tumor suppressors were

altered. This will cause cells to proliferate uncontrollably (National Cancer Institute,

2019). Furthermore, excessive hormone secretion like estrogen, was also found out to

increase the likelihood of a woman to have cervical carcinomas as well as viral

infection which was accounted to induce liver and cervical cancers (Cooper, 2000).
This information is important in order for the researchers to understand the causes of

cancer in a molecular level.

Stages of Cancer Progression. Aside from DNA mutation, Kadioglu, Seo, &

Efferth (2013) mentioned that cancer progression includes the following stages:

inflammation, cell survivability, cell proliferation, invasion, angiogenesis and

metastasis. Rudolf Virchow had already long observed that cancerous tissue produces

at area with chronic inflammation. It was because during inflammation, an

immunoresponse during microbial and allergic reactions, some of the white blood

cells produces oxygen and nitrogen species which has the ability to produce

mutagenic substance peroxynitrite aside from killing infected cells. Thus, repeated

and long term inflammation means higher chances DNA lesions nd alterations that

builds into cancerous cells (Coussens & Werb, 2002).

Unlike normal cells which had undergone DNA mutations, cancer cells has

been able to evade apoptopis. (Wong, 2011). Furthermore, cancer cells had also

exhibited cell survivability characterisitics like utilization of glutamine, some amino

and fatty acids in the absence of glucose to support aerobic respiration, as well as

destruction of slightly damaged organelles to survive nutrient deficient environment

(Boroughs & DeBerardinis, 2015).

If there is a significant difference between tumor (and cancer) cells and

normal cells, it is the tumor cells unchecked proliferation. This is due to cancer cell’s

high survivability and apoptopis avoidance. This occurs when tumor suppresors of the

cells are altered (Cooper, 2000; National Cancer Institute, 2019). But what separates

benign tumor cells from malignant/cancerous ones is the invasive ability of the latter

ones. A cancer cell dangerous cell is the ability to invade other tissues due to its loss
of ability of cell adhesion (Martin, Ye, Sanders, Lane, & Jiang, 2013) in response to

extracellular pressure set on these defective cells. It was said that cell transfer is also

common to normal cells. However, cancer and malignant tumors do not know when

to stop. Sad to say this cell invsion is the “key and trigger for cancer progression and

metastasis (Krakhmal, Zavyalova, Denisov, Vtorushin, & Peremulter, 2015).”

In order for tumor to grow, they must stimulate angiogenesis or else they

might stay to a maximum diameter of two to three millimeters. In the absence of

blood vessel growth, a tumor will remain inactive and stays on its benign stage.

Furthermore, the absence of blood vessels around the invasive tumors will prevent

them to metastasize. It (Paper , 1998; Martin, Ye, Sanders, Lane, & Jiang, 2013;

Ghulam, Lubna, Khan, Naeem, & Omer, 2014).

The final stage of cancer progression which is the characteristics of Stage IV

Cancers is the concept of metastasis or the transfer of cancer cells and their

propagation on other body parts. Big lesions due to metastasis is the cause of severe

organ lesions and ultimately, death (Krakhmal, Zavyalova, Denisov, Vtorushin, &

Peremulter, 2015; Steeg, 2016). This information is important to fully explain what

part of cancer progression Thunnus albacares bile might target in order for it to

become an anti cancer agent.

Common Treatments for Cancer. Chemotherapy, radiotherapy and surgical

operations had been the common treatments in treating cancer (Aruebo, et al., 2011;

National Institure for Cancer, 2015). Chemotherapy, the most popular approach had

stasted in the early 20th century, is the usage of one or combination of chemicals like

5-fluouracil and methotrexate to combat tumor and cancer growth. By 1960, the

introduction of adjuvant chemotherapy (follow application of chemicals to prevent

growth of secondary tumors) helped prevent the return of cancer to a patients body

(Aruebo, et al., 2011). However, chemotherapy is also known for targeting harmless

actively dividing cells like hair cells and even intestinal epithelial cells and even

uncontrollable vomiting and hair loss (Ghulam, Lubna, Khan, Naeem, & Omer,

2014).

. Meanwhile, radiation therapy or radio therapy had already started at late 19th

century thanks to the discovery of x-rays which, aside from detecting bone fractures,

had also been used in destroying cancerous tissues (Aruebo, et al., 2011) (National

Institure for Cancer, 2015). The development of computers also guided the

development of much sophisticated equipment in delivering the right amount of

radiation to tumor growth such as TomoTherapy system. However, it is noted that

radiation therapy can only target noticeable cancer or tumor growth but not minute

ones (Aruebo, et al., 2011).

The same limitations was also observed in surgeries which had also started in

late 19th century. Furthermore, this can only target solid tumors or cancer unlike

chemotherapy which has the ability to cure leukemia. However, it cannot be denied

that it could help automatic removal of cancerous tissue if possible and make the pain

caused by that cancerous tissue disappear. It also does not bring the side effects

brought upon by chemotherapy. Furthermore, the removal of the cancerous tissue in

the body had become less invasive over time, but still the type of surgery a patient

will have will still depend on the type of cancer he/she have (Aruebo, et al., 2011;

National Institure for Cancer, 2015).

Up to date, succesful cancer treatments require a combination of this methods,

especially radiation therapy and surgeries cannot target minute tumors or cancer cells.
Other methods are still available such as hyperthermia(heating up tumors to kill them

(National Institure for Cancer, 2015)), immunotherapy(introduction of antibodies

which will kill these abnormal cells) (Aruebo, et al., 2011), stem cell transplant

(works for patients with leukeia and lymphoma) (National Institure for Cancer, 2015)

and gene therapy (introduction of genetic related nucleic acids like DNA and RNA to

a target cell to correct abnormal behaviors and eradicat pathological tendency) had

been underway to become the next cure for cance (Aruebo, et al., 2011)r. This

information is relevant to further understand current cancer treatment and how

Thunnus albacares bile might fit in as anticancer agent.

Related Studies about Cancer. Solowey, et al.,( 2014) evaluated the

anticancer properties of three Israeli medicinal plants on human tumour cells. The

ethanolic extracts were applied on different cancer cell lines as well as healthy normal

cells which was later analyzed for viability, cell cycle,caspase activity, DNA

laddering analysis and Real Time PCR Analysis for mRNA related Apptopis. Results

revealed that Urtica membranacea, Artemesia monosperma, and Origanum dayi

exhibited concentration and time dependent anticancer effect via apoptopis and does

not harm normal cells,with a conclusion that these plants might possess anticancer

properties.

Liu, et al. ( 2019) determine how effective is the “novel intraoperative

chemotherapy (IOC) regimen that consists of hydroxycamptothecin, tumor necrosis

factor (TNF), 5-fluorouracil (5-FU), and calcium folinate (CF)” on the results of

patients who had undergone surgey pertaining to their colorectal cancer (CRC). Out

of 551 subjects, some of them were given post operative adjuvant (POA) therapy,

some were given with IOC and the rest got both therapies. Results revealed that IOC

increased the survival rate of patients regardless of other variables(age, sex, etc.) with
POA having the lowest survival rate. This is an indication that introoperative therapy

were enhanced and improve the chances of curing colorectal cancer.

Hyunkyoung, et al.( 2017) had evaluated the anticancer potential of a venom

taken from a jellyfish Nemopilema nomurai . Different cancer cell lines were treated

with different concentrations of venom and were analyzed using MTT Assay, Cell

Cycle analysis, Western Blot Analysis, nuclear morphology as well as Animal

Xenograph Model where the animals’ blood chemistry, Histology DNA fragmentation

of the developed tumor tissue and its immunohistological analysis were determined.

Results revealed that the venom showed cytotoxic ability on HepG2 cells without

damaging the normal cells, indicating the posibility of Nemopilema nomurai venom

as a part of cancer therapy. This information is important to determine the recent

researches regarding cancer, more specifically about cancer therapies.

Angiogenesis

Definition and Importance of Angiogenesis. It is the process involving the

proliferation, migration, and morphogenesis of endothelial cells from existing vessels

into new blood vessels (National Cancer Institute, 2018; Kadioglu, Seo, & Efferth,

2013 Paper , 1998). Angiogenesis is the term used in the formation of new blood

vessels which is significant in the process of natural growth and healing. It is also

known as a neovascularization which involves the process of “activation, adhesion,

proliferation and transmigration of endothelial cells form preexisting blood vessels”

(Paper , 1998). Also, according to Paper (1998). Angiogenesis is slow on a healthy

human and will be activated to act faster than its normal phase during wound healing

and embryogenesis. However, angiogenesis plays a role in several diseases, especially

cancer (American Society of Clinical Oncology, 2018; Kadioglu, Seo, & Efferth,
2013). According to Bielenberg and Zetter (2015), Based on the information above, it

is important to know angiogenesis because the researchers should have an idea of its

effects, what it could do, and how it could help our body.

Pathogenic angiogenesis. If control mechanism is disturbed, angiogenesis

might not stop growing, grow at a wrong place at a wrong time or might not grow at

al. This state is called pathological angiogenesis and was linked to a lot of disease.

Excessive angiogenesis was linked to disease like rheumatoid arthritis, AIDS

complications, psoriasis and diabetic retinopathy. Meanwhile, insufficient production

of blood vessel might lead to conditions like stroke, infertility, scleroderma, ulcers

and heart disease (Paper , 1998; Ghulam, Lubna, Khan, Naeem, & Omer, 2014).

Yadav, Puri, Rastogi, Satpute, & Sharma, (2015) mentioned that “tumour

angiogenesis is the proliferation of blood vessels penetrating the cancerous growth for

the supply of nutrients and oxygen’” and is vital for metastasis. As Folkman (1971)

and colleagues had observed, no tumor can grow beyond three millimeters without

angiogenesis going on. And as Ghulam, Lubna, Khan, Naeem, & Omer (2014) had

pointed out, without angiogenesis, tumor will not be malignant. When compared with

blood vessels of healthy tissues, tumor blood vessels differ in structure, are iregularly

shaped and are heterogeneous (Kadioglu, Seo, & Efferth, 2013). This information is

important to understand the diseases bought by pathological angiogenesis and provide

options regarding the scope of application of Thunnus albacares bile in the field of

medicine. Emphasis on the connections of angiogenesis with tumor development to

link angiogenesis to cancer growth to stress out the relation of this study to anticancer

products.
 Importance of anti-angiogenic products. Anti-angiogenic activities are

greatly needed to administrate because these stop or slow down the maturation of

cancer by starving it of its required blood stock (National Cancer Institute, 2018) but

it should be not that antiangiogenic do not cure cancer- it only delays its growth

(Paper , 1998). According to Zihlif, et al., (2013), the anti-angiogenic activities have

the capability to prevent the spreading of succeeding tumor at the pre-malignant stage.

Based on the study of Wang, et al., (2015), it increases the efficacy of radiotherapy,

chemotherapy, and surgical operations. But when compared with conventional

chemotherapeutic drugs, antiangiogenics are less cytotoxic, do not give out side

effects like nausea, uncontrollable vomiting, diarrhea and hair loss (Ghulam, Lubna,

Khan, Naeem, & Omer, 2014).

Methotrexate, is a well-known drug to cure chronic inflammation like

rheumatism, exhibits antiangiogenic property by inhibiting vascular endothelial

growth factors, angiopoietin 1 and 2 (Park S. , Kim, Yang, & Hur, 2011) Other

commercially available antiangiogenic which were already used in treating cancers

are the following: Bevcinuzab, Ziv-ablifercept, Suraenib, Sunitinib, and many others

(Al Abd, Alamoudi, Abdel-Naim, Neamatallah, & Ashour, 2017).

Quercetin, a form of flavonoids found fruits, is phytochemical substance

which was already proven to decrease blood vessel formation by suppressing VEGF

too. Resveratrol, found red wine was also found to be antiangiogenic as well as

rosmarinic acid (Kadioglu, Seo, & Efferth, 2013; Paper , 1998). Novastin which was

derived from shark cartilages also showed antiangiogenic potential by inhibiting beta-

Fibroblast growth factors. (Kadioglu, Seo, & Efferth, 2013)This information stresses

at out the importance of antiangiogenics as cancer therapeutics. Methotrexate was

highlighted in the list of antiangiogenic products since this was used as the standard
drug of this study. Natural angiogenic products were also presented to provide proof

that antiangiogenic products can also come from natural products, and possibly, on

Thunnus albacares bile.

Ways to Test Angiogenic Property. There are lots of methods used to

evaluate angiogenesis. There were in vivo methods like sponge implantation assay,

matrigel plug assay, zebra fish assay etc., in vitro like tube formation assay, cord

formation assay and aortic ring assay, and in ovo like Chorioallantoic Membrane

Assay (Ghulam, Lubna, Khan, Naeem, & Omer, 2014). In vivo tests are not easy to

perform and process of quantification is also difficult. However, it can fully grasp the

complex nature of angiogenesis in no way in vitro methods can achieve (Patan,

2004). In vitro methods are fast, quantifiable, more accurate and reliable (Sharkey, et

al., 2000). Chorioallantoic Membrane (CAM) Assay is generally rapid, simple and

inexpensive and was considered as precursor research in angiogenic studies. (Ghulam,

Lubna, Khan, Naeem, & Omer, 2014).

In CAM assay, fresh fertilized eggs are disinfected and incubated to a certain

number of days. Then a small volume of albumin is removed and was replaced with a

certain volume of treatments from day seven today 12 since on this stage the egg is

immunodeficient and blood vessel formation is at its fastest growth rate (Li, et al.,

2015) . The treated eggs are return to the incubator for another 24-72 hours. Then the

CAM is separated and was analyzed for further evaluation like photographically

monitoring x-y-z field of view of the CAM using a microscope and analyzing

determining the number of blood vessel in the field of view, vascular density and

blood vessel diameter (Ejaz, Seok, & Woong, 2004; Ghulam, Lubna, Khan, Naeem,

& Omer, 2014). These information is important in order for the researchers to be

aware of the possible methods in testing a substance antiangiogenic activity. CAM

assay were given emphasis since this was the method chosen by the researcher in

determining Thunnus albacares bile antiangiogenic potential.

Related Studies on Angiogenesis. Lee Shukla, Kim, and Kim,(2015) studied

Nelumbo nucifera’s ability as an anti-angiogenic agent via in vivo and in vitro

angiogenic testing methods. In utilizing the in vivo technique, they used chick

chorioallantoic membrane assay and the proliferation, viability, and capillary tube

formulation in human umbilical vein endothelial cell (HUVEC). Results revealed that

the said plant inhibited antiangiogenic potential by inhibiting the VEGF.

Abdolmaleki, Ali Arab, Amanpour, and Muhammad nejad (2016) determined

the anti-angiogenic activity of the ethanolic extract of Artemesia sieberi using in vivo

and in vitro methods. The extracts of A. sieberi were tested using both chick

chorioallantoic membrane (CAM) assay for the in vivo model and three-dimensional

angiogenesis assay or in vitro model. The extracts were inspected in the human

umbilical vein endothelial cells and results revealed that the ethanolic extract of A.

seiberi is strongly capable to block angiogenesis in CAM and HUEVEC assay. Both

the extracts showed inhibitory activities towards VEGF included proliferation, tube

formation, and CAM angiogenesis in vivo. The extracts of A. sieberi strongly

interfered with the angiogenesis in the HUVEC and in the CAM assay.

Marsdenia tenacissima angiogenic effect was evaluated by Huang (2013).

They utilized the in vivo model while the researchers worked on human umbilical vein

endothelial cell (HUVEC), proliferation, migration, and capillary-like membrane tube

formation for the in vitro tests. Results showed that the extract of M. tenacissima

hinders the proliferation of HUVEC’s by stopping the cell cycle advancement from

G1 to S. It also stops the tube formation and migration in of HUVEC’s. Furthermore,

M. tenacissima extract is capable in the decrease of the developmental of blood

vessels in the chick embryo based on the results of the CAM assay conducted before

hand. Therefore, the collected details proved that the said plant can inhibit the growth

of angiogenesis. This information gives an idea on how other researchers conducted

their studies and this also aims to know the methods used and results gathered by the

researchers.

Anti-oxidative Property

Definition and Importance? Anti-oxidants are substances that may be

natural, like from fruits and vegetables, or artificially made that slow down or prevent

cell for further damage (U.S. National Library of Medicine, 2018). Damage in cells

can be caused by radicals that our body produces under stress, environmental and

other pressures (Maeturana, 2017). They are sometimes called “free-radicals

scavengers” (Ware, 2018).

Free radicals are molecules that are considered to be very unstable. Because of

its unreliability, it tends to attract electron from other molecules which causes future

cell damage or disease. If free radicals continue to attract electrons from other

molecules to attain stability, it could cause a ripple effect and could lead to dangerous

diseases like cancer, Alzheimer’s disease etc. Anti-oxidants are substances that can

neutralize free radicals in the form of giving off electrons so that they will not affect

other molecules. It is important to learn things about what anti-oxidant is because

everybody should know what effects it could do to our body. It gives us an idea on

how anti-oxidants help our body. Upon conducting such tests on anti-oxidants,

discovery of substances that can provide prevention or cure for cellular diseases will

be done by the researchers.

 Examples of antioxidants. Beta carotene, Vitamin A (retinol), Vitamin C

(ascorbic acid) and Vitamin E (Alpha-Tocopherol) were examples of antioxidants

which can be found on the food we eat. Beta carotene decreases free radical damage,

and its ability to quench singlet oxygen. Vitamin A which can be produced from Beta

carotene acts as an antioxidant by maintaining epithelial tissues and glands. Vitamin

C role is due to hydroxylation and chemo preventive properties. It also scavenges

oxygen and nitrogen species. (Birangane, Chole, Reddy, & Shivajii, 2011) Meanwhile

Vitamin E, is an effective antioxidants specially that of hydroperoxide. It is protective

towards lipid peroxidation, improves T cell mediated response. It was considered as

the most important lipophilic, radical scavenging antioxidant which could help avoid

liver diseases and atherosclerosis brought upon by oxidative stress (Birangane, Chole,

Reddy, & Shivajii, 2011) (Niki, 2015). This information is important to be aware of

common antioxidants with emphasis with Vitamin E since this was the standard drug

used where the antioxidative property of Thunnus albacares bile was compared.

Ways to test anti-oxidative ability. The different assays which can be used to

test anti-oxidative properties of different substances are ABTS assay, FRAP assay,

ORAC assay, and DPPH assay (Moharram, and Youseff, 2014) and all of them are in

vitro methods, which are known to distinguish anti-oxidant properties but it may be

difficult on concerning cellular uptake and transport process because in vitro method

can be hard to apply to biological systems (Alam, and Rafiquzzaman, 2013).

Examples of in vivo methods are ferric reducing ability in plasma, catalase (CAT) and

lipid peroxidation (LPO) Assay. (Alam, Bristi, & Rafiquzzaman, 2013).

Alpha (α), α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging

method was the first method used to determine antioxidative property of a substance

(Kedare & Singh, 2011). It is known as the simplest method and its concept revolves
around the property of a stable free radical known as α, α-diphenyl-β-picrylhydrazyl

or better known as DPPH. DPPH is violet in color and produces and absorption band

in ethanol at 517nanometers, but when a substance donates an electron unto it, it

becomes colorless. (Alam, Bristi, & Rafiquzzaman, 2013)

Marth Lewis (2012) devised a way of testing antioxidative property of crude

extracts with minimal amount of extract and DPPH needed. In her procedure, she

utilized α-tocopherol as the positive control. Tris-HCl buffer solution was also used.

The experimental treatments concentration were prepared in the cuvettes (0.2, 0.15

and 0.1mg/mL) together with negative control (solvent) and positive control. In each

cuvette, on milliliter of 0.1millimolar of DPPH solution was placed. The cuvettes

were set aside in a dark area for 30 minutes and each solutions’ absorbance value was

read using a spectrophotometer set at 517 nanometers. The absorbance values of each

data is converted into percentage using the formula:

DPPH scavenging effect (%) = [(AO - A1)/ AO] × 100

Where AO was the absorbance of the control and A1 was the absorbance of the

sample (Lewis, 2012).Knowing the different methods used in determining anti-

oxidative property these give the researchers an idea on how to test antioxidative

activities of different substances. Emphasis on DPPH assay was given since this is the

procedure used to test the antioxidative property of Thunnus albacares bile.

Related Studies on Antioxidants. The anti-oxidative effect of

Tabernaemontana pandacaqui leaf extract was tested using DPPH radical scavenging

assay. Tabernaemontana pandacaqui leaves ethanolically extracted and was assayed

via DPPH radical scavenging assay. Results revealed that the plant extract yielded

high antioxidative ability (Samar, et al., 2018).

 Mandade, Sreenivas, & Choudhury(2011) evaluated the free radical

scavenging and antioxidative ability of Carthamus tinctorius extracts. Aerial part of

the plant were soaked in aqueous –methanol and undergone solvent extraction. Its

antioxidative ability were evaluated using ferric thiocyanate method, hydrogen

peroxide scavenging assay, dpph radical scavenging assay, ABTS radical cation

decolorization assay, and Superoxide anion radical scavenging activity assay with α-

tocopherol as the standard drug. It was concluded that the plant extract do possessed

antioxidative and radical scavenging ability.

Anti-oxidative properties are also found on herbs and spices. The results were

determined by liquid-phase extraction, solid-phase extraction, super-critical fluid

extraction, and 2, 2-diphenyl-1-picryl-hydrazyl-hydrate or DPPH method. Maca or

Lepidium meyenii was studied by researchers about its association with anti-oxidants,

alkaloids, and phenols. Using the FRAP assay, other metabolites were also measured

such as ferric reducing anti-oxidant potential (FRAP), hydroxyl radical scavenging

ability (HRSA), lipid-peroxidation contents (TPCs), total alkaloid contents (TACs),

and total sterol contents (TSCs). The solvent picked to be tested with was chloroform.

The outcome showed that TPCs association to LPIA and FRAP was (P<0.05) and

TACs association with HRSA, LPIA, and FRAP was (P<0.05). It states that alkaloids’

anti-oxidative effect was greater than phenols. Also the two components were a

problem for Maca’s anti-oxidation to be the most significant chemical (Yahin, et al.,

2017). These information helped the researchers on what method to use in testing the

anti-oxidative ability of Thunnus albacares bile.

Thunnus albacares
 Taxonomic Classification of Thunnus albacares. The taxonomic

classification of yellow fin tuna is as follows:

Kingdom: Animalia

Phylum: Chordata,

Class: Actinopterygii,

Order; Perciformes,

Family: Scombridae

Genus: Thunnus

Species: albacares

It is formerly known as Scomber albacares in the year 1788 (World Register

of Marine Species, 2018; IUUCN Red List, 2018; IT IS Report, 2018).

Distribution of Thunnus albacares. Thunnus albacares is located in the

waters worldwide, specifically in the tropical and subtropical waters (Florida Museum

of National History Gainesville, 2017). They are all over the world because they tend

to travel or migrate. Some patterns of their migration come from the U.S Pacific Coast

to Japan (MarineBio, 2013). It is also said they can be found in the Canaries, Mediera,

Azores and the Bay of Spain by the researchers (Bonnaterre, 1788). Thunnus

albacares is a very widespread species of tuna that could be found anywhere but they

seem to be absent in the Mediterranean Sea. The significance of finding the

whereabouts of it is because it is needed by the researchers to locate the places where

Thunnus albacares can easily be found for the study.

Morphological Structure of Thunnus albacares. Thunnus albacores, more

recognized as yellowfin tuna, is almost like a big “torpedo-shaped” tuna. Thunnus

albacares posses a circular body which is colored metallic blue green at the top and

silvery white beneath it, with many vertical lines. Thunnus albacares is a well known

commercial sea creature that weighs from 400 pounds till 880 pounds. This fish seems

to be a sight-oriented predator because of its predating habit at surface water during

the day (Gardieff., 2017). This sea creature is also the second extremely significant

tuna species in the Philippines based on its catch rate (Pedroso-Gerasmio, 2012). The

scales minute is about 270 in lateral line. Its air bladder is present. Its pectorals are

long, reaching to or over the beginning of the second dorsal excluding in some really

huge specimens. Second dorsal and anal are often much elongated in large specimens

with no venules (striations) on the surface of the liver, the left lobe of which is

sometimes divided in two, and the right lobe of which is longer than the other. Its air

bladder is narrow and lengthy; it is not divided at the anterior end. Dorsal fin has 13

or 14 spines. It has eight to 11 dorsal finlets; eight to ten anal finlets, and Gill rakers

seven to 11 + 19 to 22 (Schaefer, et al, 1963). It is very noticeable that some of the

informations gathered were mostly very old because there are not that many recent

studies made about Thunnus albacares. The information is important because it tells

us a lot about the body structure of Thunnus albacores which is important to this

study.

Related Studies about Thunnus albacares. Yellowfin Tuna in the

southwestern coasts of Taiwan is believed to be acting differently from other yellow

fin tuna in other seas. It is said that its mean swimming depth at daytime reaches 74.4

meters (± 50.7 meters) while during night it manages to reach 94 meters (± 72.5

meters). Other than its mean swimming depth, its maximum diving depth also reaches

to 1000 meters at four degrees Celsius. Vertical movements of yellowfin tuna from

Taiwan were discovered by the researchers. Majority of their vertical movements is

50-meters deep and also it ranges in mixed layers. The discoveries were known

because of pop-up satellite archival tags used to track and observe seven out of eleven

yellowfin tunas released in these coasts (Jinn-ShingWeng, et al., 2017). From coasts

of Taiwan to the Pacific Ocean specifically on the Western, Eastern, and Central part,

some facts about the population and management of tuna were determined by the

researchers. Out of the three parts in the Pacific, one out of three was nearly 100%

heterogeneous population. A specific method which is “double-digest restriction site

associated DNA using Next Generation Sequencing Technology” was used by the

researchers. “Fishery Independent Verification” was upholding yellowfin tuna

because of the results (Grewe, 2015). Yellowfin tunas from the South Africa (SAF)

fisheries were tested via genomic analysis because of lack of knowledge about

recruitment of yellowfin tuna “spawning population” from Atlantic and Indian Ocean.

The researchers discovered that there’s no yellow fin tuna inhabiting the Atlantic

Ocean but the tunas that migrated from the Indian Ocean were recorded to have been

seen here. Thunnus albacares from Indian Ocean migrated to the Atlantic spawning

site that caused the “mixed-stock fishery” in the Atlantic (Mullins, et al., 2018). This

aims to understand deeper what Thunnus albacares is.

Bile

Definition and Purpose of Bile. It is a complicated body fluid chemical that

assists during the “digestive” cycle where it is being kept at the gallbladder after it is

freed and produced at the liver. This complex substance breaks down fats for it to

become fatty acids which flow throughout the body by the digestive path (Chervona,

2018). Bile is a fluid which is composed mainly of bile acids that helps the crucial

process of “digestion”, and “absorption” of fat-soluble vitamins, fats, and a bunch of

body feces which is removed by the secretion into the bile and body trash produced
deposition (American Accreditation Health Care Commission, 2016). The information

about bile and its chemical composition is significantly important in knowing what a

complex body fluid could do in providing future ideas and guidelines on what it could

possibly use for.

Chemical Components of Bile. Boyer (2014), found a chemical composition

that makes up the fish bile like bile salts, bilirubin, biliverdin, phospholipid, amino

acids, enzymes, steroids, cholesterol, porphyrins, vitamins and heavy metals,

exogenous drugs, xenobiotics, and environmental toxins. Other than that, bile is

composed of phospholipids, cholesterol, pigments, water and electrolyte chemicals

(Encyclopedia Britannica, 2018). This aims to find helpful or harmful chemicals

present in fish bile that may be dangerous or useful for the researchers or other

people.

How to extract fish bile? Researchers buy excess gall bladder in the market

and then air it is dried to remove its moisture. Next, the bile is extracted through

methanol soaking (Enrile, Lim, and Go, 2018). According to the study of Mathew, et

al. (1986), to extract the bile, gall bladders were accumulated by opening the stomach

and parting the gall bladder without causing damage to the bile sack. After getting the

gall bladder, it must be soaked into water to remove blood and other contaminants,

and then the bile was gathered by pricking the gall bladder with a needle and oozing it

to take its extract. The extracts were reconstituted in 250 micro liter of methanol and

can be analyzed by liquid chromatography-tandem mass spectrometry (Ros, et al.,

2015). Meanwhile, Enrile, Lim, & Go, (2012), obtained their Chanos chanos bile

through methanolic soaking. This information gives the researchers options on how to

extract bile form Thunnus albacares.

 Bile and angiogenesis. Enrile, Lim, & Go, (2012) study revealed that

Chanos chanos bile exhibits antiangiogenic properties. Park K. , et al., (2007) also

had the same results when they tested bile acid acylated heparin derivative using

capillary-like tube formation assay and Matrigel plug assay. It is worth noting that

heparin, a famous anticoagulant, is naturally produced in our body by liver (National

Cancer Institute, 2019). Presta, et al.( 2003) had pointed out that heparin derivatives

are angiostatic in nature. Furthermore, Dao, et al.( 2018) proved that heparin

VascularEndothelial Growth Factors, interfering in blood vessel growth in lung tissue

in vitro and in vivo. However, Kundu, et al., (2017) studies on bile acids revealed

that human hydrophilic bile acids chenodeoxycholic acid (CDCA) exhibited

proangiogenesis. Maybe, it could be accounted it to the type of substance found in

lungs the one that spelled out the difference. This information is important to explain

what specific substances might probably explain Thunnus albacares bile’s

antiangiogenic activity.

Bile and Antioxidants. Daiber & Daiber( 2012) had pointed out that bilirubin

and biliverdin, a component of bile, possess antioxidative properties. Furthermore, it

was reported that bile salts micelles possesses antioxidative properties when tested

using chemiluminescent assay (Roda, et al., 1998). Bile acids were also known as

antioxidants by scavenging of peroxyl radicals by direct oxidation of the bile acids

(Delange & Glazer, 1990). This information is important to explain what is in the bile

that might possess antioxidative properties.

Related Studies on Bile. Polycyclic Aromatic Hydrocarbons (PAHs) was

believed to be present on the fishes of Morocco’s seas. PAH’s studies were less than

other published topic, so the study of bile metabolites of PAHs to fishes from open

sea and the lagoon to differentiate the levels of PAH metabolites in different species
of fishes was conducted by the researchers. Fortunately, the results were 1

hydroxypene (<LOD-15.56 mg/ml) bile metabolites that was present in fishes. There

was also a lower concentration of 1 hydroxypenanthrene (<LOD-9.6 mg/ml). This

means that the concentrations of the results of these studies were lower than other

studies before (Baali, et al., 2016).

On the other hand, lecithin and other bile components were tested to find out

if it is related to diet intestinal inflammation in Atlantic salmon. Unfortunately, the

results were not good and the test was also a bit dangerous because it indicated

negative effects (Korther, et al, 2016). Fish bile is also known for being an olfactory

stimulus in fishes, so the study was conducted using an extracellular

electrophysiological technique to determine the level of its stimuli effect. Testing for

bile salts, it was concluded that fish bile is highly effective as facial taste stimuli

(Rolen, and Carpio, 2008).

Vedagopuram, et al. ( 2013) synthesized bile acids to form bile acid tamoxifen

conjugates from lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid

(CA). which shoes high cytotoxicity against cancer cell lines and on in vivo studies

using 4T1 tumor models. It was sid that the bile derivatives induce apoptopis and

arrest cells in sub Go stages.

Enrile, Lim, & Go (2012) determined the anti-angiogenic and antibacterial

property of Chanos chanos bile using CAM Assay and Agar Well Diffusion Method,

respectively. Bile was obtained using methanolic extraction method. Results showed

that there is a significant decrease in blood vessels on CAM as treated with

experimental treatments and the experimental treatments produced zone of inhibition

against Staphylococcus aureus and Escerichia coli which is comparable to

Amoxicillin.

This related studies had been become a basis in conducting the researchers’

study, especially that of (Enrile, Lim, & Go, 2012).

Al Abd, A., Alamoudi, A. J., Abdel-Naim, A. B., Neamatallah, T. A., & Ashour, O.
M. (2017). Anti-angiogenic agents for the treatment of solid tumors: Potential
pathways, therapy and current strategies – A review. Journal of Advanced
Research, 591-605.

Alam, M., Bristi, N., & Rafiquzzaman, M. (2013). Review on in vivo and in vitro
methods evaluation. Saudi Pharmaceutical Journal , 143-152.

Ames, B. N., Swirsky-Gold, L., & Willet, W. C. (1995). The Causes and Prevention
of Cancer. Proc. Natl. Acad. Sci. USA, 5258-5265.

Aruebo, M., Vilaboa, N., Saez-Guitierez, B., Lambea, J., Alejandro, T., Valladares,
M., & Gonzales-Fernandez, A. (2011). Assessment of the Evolution of Cancer
Treatment Therapies. Cancers(Basel), 3279-3330.

Birangane, R., Chole, D., Reddy, K., & Shivajii. (2011). A Review of Antioxidants.
Journal of Indian Academy of Oral Medicine andRadiology, 351-353.

Boroughs, L. K., & DeBerardinis, R. J. (2015). Metabolic pathways promoting cancer

cell survival and growth. Nature Cell Biology, 351-359.

Cooper, G. (2000). The Cell: A Molecular Approach. 2nd Edition. Sunderland MA:
Sinauer Associates.

Coussens, L. M., & Werb, Z. (2002). Inflammation and cancer. Nature(420)6907,

860-867.

Daiber, J., & Daiber, A. (2012). Direct antioxidant properties of bilirubin and
biliverdin. Is there a role for biliverdin reductase? Frontiers in Pharmacology.

Dao, D., Anez-Bustillos, L., Ourieff, J., Pan, A., Kishikawa, H., Fell, G., . . . Puder,
M. (2018). Heparin impairs angiogenic signaling and compensatory lung
growth after left pneumonectomy. Angiogenesis, 837-848.

Delange, R., & Glazer, A. (1990). Bile acids: antioxidants or enhancers of

peroxidation depending on lipid concentration. Archives of Biochemistry and
Biophysics, 19-25.
Ejaz, s., Seok, K., & Woong, L. (2004). A novel image system for precise
quantification of angiogenesis. Tumori, 611-617.

Enrile, J. O., Lim, J., & Go, L. (2012). Anti-angiogenic and Antibacterial Property of
Chanos chanos Bile. Retrieved from Scribd:
https://www.scribd.com/document/182747806/ANTI-ANGIOGENIC-AND-
ANTIBACTERIAL-PROPERTY-OF-BANGUS-BILE-EXTENDED-
ABSTRACT-doc

Folkman, J. (1971). Antiangiogenesis: New Concept for Therapy of Tumor. Annals of

Surgery, 409-416.

Ghulam, J., Lubna, S., Khan, S., Naeem, H., & Omer, M. (2014). Assessment
methods of angiogenesis and Present Approaches for Its Quantification.
Cancer research Journal Vol 2, No. 3, 47-62.

Hyunkyoung, L., Seong Kyeong, B., Munki, K., Min, J., Minkyung, K., Sujeoung, Y.,
. . . Kim, E. (2017). Anticancer Effect of Nemopilema nomurai Jellyfish
Venom on HepG2 Cells and a Tumor Xenograft Animal Model. Evidence-
Based Complementary and Alternative Medicine.

Kadioglu, O., Seo, E., & Efferth, T. (2013). Targeting Angiogenesis by

Phytochemicals. Medical Aromatic Plants.

Kedare, S. B., & Singh, R. (2011). Genesis and development of DPPH method of
antioxidant assay. Journal of food Science Technology, 412-422.

Krakhmal, N. V., Zavyalova, M. V., Denisov, E. V., Vtorushin, S. V., & Peremulter,
V. M. (2015). Cancer Invasion: Patterns and Mechanisms. Acta Naturae, 17-
28.

Kundu, S., Bansal, S., Muthukumarasamy, K., Sachidanandan, C., Motiani, R. K., &
Bajaj, A. (2017). Deciphering the role of hydrophobic and hydrophilic bile
acids in angiogenesis using in vitro and in vivo model systems.
MedChemComm 8(12), 2248-2257.

Lewis, M. (2012, November 5). Natural Product Screening Anti Oxidant Screen
DPPH of Extract Crude Extract1. Retrieved from SCRIBD:
https://www.scribd.com/document/271054618/15-3b-Natural-Product-
Screening-Anti-Oxidant-Screen-DPPH-of-Extract-Crude-Extract1

Li, M., Pathak, R., Lopez-Rivera, E., Friedman , S., Aguirre-Ghiso, J., & Sikora, A.
(2015). The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an
Efficient Xenograft Model of Hepatocellular Carcinoma. Journal of
Visualized Experiments.
Liu, Z., Zou, Y., Rong, Y., Li, C., Tian, Y., Lin, H., . . . Zhao, X. (2019).
Intraoperative Chemotherapy with a Novel Regimen Improved the
Therapeutic Outcomes of Colorectal Cancer. Journal of Cancer, 5986-5991.

Mandade, R., Sreenivas, S., & Choudhury, A. (2011). Radical Scavenging and
Antioxidant Activity of Carthamous tinctorus Extract. Free Radicals and
Antioxidants.

Martin, T. E., Ye, L., Sanders, A. J., Lane, J., & Jiang, W. G. (2013). Cancer Invasion
and Metastasis: Molecular and Cellular Perspective. Madame Curie Biosciene
Database.

National Cancer Institute. (2019, August 1). Definition of Cancer. Retrieved from
NCI Dictionary of Cancer Terms:
https://www.cancer.gov/publications/dictionaries/cancer-terms/def/cancer

National Cancer Institute. (2019, October 17). NCI Dictionary of Cancer Terms.
Retrieved from National Cancer Institute:
https://www.cancer.gov/publications/dictionaries/cancer-terms/def/heparin

National Institure for Cancer. (2015, April 29). Types of Cancer Treatment. Retrieved
from National Institute for Cancer:
https://www.cancer.gov/about-cancer/treatment/types

Niki, E. (2015). Evidence for beneficial effects of vitamin E. Korean Journal of

Internal Medicine, 571-579.

Paper , D. H. (1998). Natural Products as Angiogenesis Inhibitors. Planta Medica,

686-695.

Park, K., Kim, Y., Nam, J., Lee, S., Park, R., Kim, S., . . . Byun, Y. (2007).
Antiangiogenic effect of bile acid acylated heparin derivative. Pharmaceutical
Research, 176-185.

Park, S., Kim, H., Yang, Y., & Hur, D. (2011). Effects of methotrexate on vascular
endothelial growth factor, angiopoietin 1, and angiopoietin 2 in nasal polyps.
American Journal of Rhinology and Allergy, 129-132.

Patan, S. (2004). Vasculogenesis and angiogenesis. Cancer Treatment Research, 3-

32.

Presta, M., Leali, D., Stabile, H., Ronca, R., Camozzi, M., Coco, L., . . . Rusnati, M.
(2003). Heparin derivatives as angiogenesis inhibitors. Current
Phamaceutical Design, 553-66.

Roda, A., Ruso, C., Pasini, P., Piazza, F., Feroci , C., Kricka, L., & Baraldini , M.
(1998). Antioxidant properties of bile salt micelles evaluated with different
 chemiluminescent assays: a possible physiological role. Journal of
Bioluminescence and Chemoluminescence, 327-337.

Sharkey, A., McPherson , S., Day, K., McPherson, A., Malik, S., Licence, D., . . .
Charnock-Jones, D. (2000). Vascular endothelial growth factor expression in
human endometrium is regulated by hypoxia. Journal of Clinical Endocrinol
Metabolism, 402-409.

Shiel, W. C. (2018, December 4). Medical Definition of Cancer. Retrieved from

Medicine Net: https://www.medicinenet.com/script/main/art.asp?
articlekey=2580

Solowey, E., Lichenstein, M., Sallon, S., Paavilainen, H., Solowey, E., &
Lorberbourn-Galski, H. (2014). Evaluating Medicinal Plants for Anticancer
activity. The Scientific world Journal. Retrieved from The .

Steeg, P. D. (2016). Targeting metastasis. Nature Reviews Cancer, 201-218.

The Global Cancer Observatory. (2019, May). Philippine Fact Sheets. Retrieved from
International Agency for Cancer Research:
https://gco.iarc.fr/today/data/factsheets/populations/608-philippines-fact-
sheets.pdf

Vedagopuram, S., Bansal, S., Motiani, R. K., Kundu, S., Muppu, S., Majumdar,
T., . . . Bajaj, A. (2013). Design, Synthesis, and Mechanistic Investigations of
Bile Acid–Tamoxifen Conjugates for Breast Cancer Therapy. Bioconjugate
Chemistry, 1468-1484.

Wong, R. S. (2011). Apoptosis in cancer: from pathogenesis to treatment. Journal of

Experimental and Clinical Research, 87.

World Cancer Research Fund. (2019). Worlwide Cancer Data. Retrieved from World
Cancer Research Fund: https://www.wcrf.org/int/about-us/about-site/copyright

Yadav, L., Puri, N., Rastogi, V., Satpute, P., & Sharma, V. (2015). Tumour
Angiogenesis and Angiogenic Inhibitors: A Review. Journal of Clinical and
Diagnostic Research, XE01-XE05.