ANTIANGIOGENIC ACTIVITY OF GAMBHARI (Gmelina arborea)

ON MALLARD (Anas platyrhynchos) DUCK EMBRYO

JEANNE KELLY L. TIMBAL

GUEN MICAH B. TAMAYO
CYRIL SHANE B. CONOL

A RESEARCH PROPOSAL SUBMITTED IN PARTIAL FULLFILLMENT FOR THE

REQUIREMENT IN THE SUBJECT RESEARCH III
(SCIENCE AND TECHNOLOGY)

MR. JADE B. MONTEJO

RESEARCH III TEACHER

SPECIAL SCIENCE PROGRAM (SSP)

VALENCIA NATIONAL HIGH SCHOOL

OCTOBER 2019
 INTRODUCTION

Background of the Study

The use of medicinal plants is widely used by human population, especially in

developing countries, where the lack of financial resources hinders access to the treatment

of diseases by conventional medicine. Medicinal plants among indigenous people

throughout the world especially in the Philippines, has been a prevalent treatment for

illnesses because of its natural and organic components. Also, it is capable of treating

infections and several diseases because of its natural products derived from plants for

treatment. This also manifest that nature is a golden symbol to demonstrate the relationship

between man and environment.

Cancer is one of the most common disease that is very alarming to each and every

one throughout the world because of its serious potential life-threatening illness. The

disease is also thought untreatable, unbearably painful and is incurable that leads a person

to death.

Angiogenesis is the production of new blood vessels. The process involves the

migration, growth, and differentiation of endothelial cells, which line the inside wall of

blood vessels. Angiogenesis follows a series of steps involving endothelial cell activation

and degradation of the basement membrane, accompanied by endothelial cell migration,

proliferation and pipe formation (Jadav, Mane, and Kanase, 2011). Angiogenesis has

become one of the big issues in fighting against the progress on inhibiting cancer. At

present, an ongoing investigation of plant extracts which has the potential to inhibit

angiogenesis, takes place.

 Gmelina arborea, or commonly known as Gambhari, is a plant that grows

throughout the community because of its abundant classification, and is demonstrated its

role in traditional medicine according to Rajan, Sarumathy, and Sudha (2011).

Despite of the properties of the plant, the researchers were motivated to produce an

organic complementary remedy that would potentially contain the major components to

reduce the issues in cancer.

The aforementioned motivated the researchers to conduct the study on the

antiangiogenic activity of Gambhari (Gmelina arborea) specifically on its fruit exocarp,

leaf and root extract to serve as a new path as alternative treatment to cancer.

Statement of the Problem

This study aims to investigate the antiangiogenic activity of Gambhari (Gmelina

arborea) fruit exocarp, leaf, and root extract using Mallard (Anas platyrhynchos) duck

embryo.

Specifically, it sought to answer the following questions:

1. what are the bioactive compounds present in the G. arborea fruit exocarp, leaf

and root on A. platyrhynchos?

2. how to examine the blood vessel formation of A. platyrhynchos embryo with the

three (3) different treatment?

3. is there any significant effect of the three (3) different treatments of G. arborea

on A. platyrhynchos in contrast to the formation of cancer?

4. which of the three (3) treatments from the different parts of G. arborea plant is

the most effective in _______________________ of the A. platyrhynchos?

 Objectives of the Study

This study aims to investigate the antiangiogenic activity of Gambhari (Gmelina

arborea) fruit exocarp, leaf, and root extract using Mallard (Anas platyrhynchos) duck

embryo.

Specifically, the study aims to:

1. identify the bioactive compounds present in the G. arborea fruit exocarp,

leaf and root.

2. examine the blood vessel formation of A. platyrhynchos embryo with the

three (3) different treatments.

3. find out the significant effect of the three (3) different treatments of G.

arborea on A. platyrhynchos in contrast to the formation of cancer.

4. determine which of the three (3) treatments from the different parts of G.

arborea plant is the most effective in _______________________ of the A.

platyrhynchos.

Significance of the Study

This study is an assessment on the antiangiogenic activity of Gambhari (Gmelina

arborea) fruit exocarp, leaf, and root extract using Mallard (Anas platyrhynchos) duck

embryo. The study will contribute to the community especially in the medical field

regarding on the rapid increase of cancer fatality in the country. This study will also benefit
the researchers who are concerned in this field of medicine and to the pharmacists for the

production of drugs that can aid the cure of abnormally growing cells of the body which

might lead to cancer formation.

The output of this research will serve as a significant baseline data in generating

more antiangiogenic pathways and antiangiogenic progress for producing a remedial drug

from an organic sample and will also benefit to the people who are in risk of having cancer.

Moreover, this study is significant in finding much potent alternative treatment as a

cancer-fighting agent combined with additional remedy.

Finally, this paper will serve as a significant baseline for reference to the future

researchers that are discovering and wants to know more about angiogenic inhibition using

organic remedy.

Scope and Limitations

This study is limited only on the antiangiogenic activity of Gambhari (Gmelina

arborea) fruit exocarp, leaf, and root extract that can inhibit angiogenic signaling pathway.

It also evaluates the antiangiogenic components of Gambhari (Gmelina arborea)

specifically on its fruit exocarp, leaf, and root extract. The study also aims to determine the

blood vessel formation of Mallard (Anas platyrhynchos) duck embryo by the different

concentrations used and compare the significant difference between the three treatments of

Gambhari (Gmelina arborea) used to the eggs.

The study will be conducted at the Valencia National High School on the months of

March to September 2020.

 REVIEW OF RELATED LITERATURE

Angiogenesis

Angiogenesis is the development of the current vasculature in the blood vessels. In

both health and disease, it occurs during life, beginning in utero and continuing to old age.

No metabolic tissue in the body exceeds a few hundred micrometers from a capillary blood

formed through the angiogenesis process. Changes in metabolic activity contribute to

proportional angiogenesis changes and therefore proportional capillarity changes. In this

regulation, oxygen plays a crucial role. Hemodynamic considerations are important for

vascular network survival and vessel wall structural changes.

Recognition of the possible therapeutic value of angiogenesis regulation has stimulated gre

at interest over the past 40 years. It can be therapeutic in cancer, ophthalmic disorders,

rheumatoid arthritis, and other diseases to decrease or stop angiogenesis. Depending on

functional requirements, capillaries expand and regress in healthy tissues. During weight

gain, capillaries grow in adipose tissue and regress during weight loss. Evidently, in life,

angiogenesis occurs. (Adair and Montani et al., 2010)

Angiogenesis on Health

Some natural health products that inhibit angiogenesis also display certain cancer-

related behaviors. Cancers without angiogenesis stay asleep. Rapid logarithmic growth

following the creation of a blood supply. The angiogenic change appears to be activated

when the balance switches from angiogenic inhibitors to angiogenic stimulators. Natural
health products contain a variety of complex organic chemicals with synergistic activity.

They can inhibit angiogenesis by dealing with multiple pathways and other activities that

can impact cell signaling, the apoptotic pathway, and the interaction between cancer cells

of both the immune system. (Sagar, Yance and Wong et al., 2006)

Angiogenesis on Blood Vessel

New activation of the blood vessel gives tumor survival advantage. Cell survival

and growth depends on a sufficient supply of oxygen and nutrients as well as harmful

substances being removed. Endothelial cells obtain a stimulating sign from angiokinins in

the natural and orderly formation of new blood vessels and secrete special enzymes such as

matrix metalloproteinase (MMP) and heparinize that end result in extracellular matrix

(ECM) dissolution. The close junctions of the endothelial cell are broken. The endothelial

cells can then project through the newly established spaces and arrange new capillary tubes

to the source of the blood. (Sagar, Yance and Wong et al., 2006)

Angiogenesis on Cancer

Angiogenesis plays a critical role in cancer growth because if solid tumors are to gr

ow beyond a few millimeters in size, they need a blood supply. Cancer is a major health

issue in developed countries where it is the second often associated cause of death with

population aging and lifestyle. (Urruticoechea et al., 2010) For growth and metastasis,

cancer cells require access to blood vessels. The discovery of angiogenic inhibitors offers

hope that carcinomas can reduce mortality and morbidity. (Cobleigh, 2003)

Angiogenesis Inhibitors
 Angiogenesis inhibitors are either classified as immediate inhibitors in expanding

vasculature influencing endothelial cells or as Lookman (2012) backhanded inhibitors

averting articulation or blocking angiogenesis inducers action. Direct endogenous

angiogenesis inhibitors, for example, angiostatin, endostatin, arrestine, canstatin, tumstatin

and others are parts discharged from various ECM particles on proteolysis. Different

angiogenesis inhibitors are designed to attack endothelial vascular cells and suppress

angiogenesis of the tumor. Targeting cells that promote tumor growth rather than cancer

cells themselves is a relatively new approach to cancer therapy that is especially promising

as these cells are genetically stable and therefore less likely to produce mutations that cause

them to quickly develop drug resistance. (Kerbel and Folkman et al., 2002)

Gmelina arborea

Gmelina arborea is a fast-growing, unarmed, and medium to large deciduous tree

with a broad, spreading canopy with numerous branches forming a huge, shady crown. It

can develop from 3 to 30 meters tall, sometimes even tallest. The straight, cylindrical bole

is usually about 50 cm in diameter, but specimens up to 140 cm have been recorded. It

could be unplugged for 6-10 meters. The tree is harvested from the wild for local use as

meat, food and medical supplies. The wood is of very good quality, it is used widely and is

often traded. The tree has suitable characteristics for agroforestry, with rapid growth, ease

of establishment and relative freedom from insects outside its native range (can be searched

repeatedly without damage). It is a particularly promising fuel wood plant, because it can

be easily developed, regenerates well from both sprouts and seeds, and grows quickly. It

has been implemented as a plantation crop in many countries, and large plantations are
located in South East Asia, West Africa and South America. It is also sometimes cultivated

as a decorative, placed as an avenue tree. (Fern, 2019)

Gmelina arborea Leaves

Leaves discolor, 10–25 about 7.5–18 cm, wide-ovate or ovate-cordate, accuminate

or caudate at the apex, subordinate to rounded or truncate with 2 glands at the base.

Complete, cracked or lobbed on turions or young plants, sparsely to thick lepidote and

glaucous-green and glabrous to velvety stellate-pubescent or -tomentose below; 4–5 lateral

nerves on each side of the midrib, the lowest basal pair; 5–15 cm long petiole (Zambesiaca,

and Fernandes, et al., 2005). The bark, leaves and roots contain traces of alkaloids and are

used medicinally in native plants. As a blood purifier, laxative, stomach, tonic, and venom

cure, the roots have a high medicinal value. The leaf sap is used as a demulcent in the

treatment of gonorrhoea and cough, as well as in the treatment of wounds and ulcers. (Fern,

2014)

Gmelina arborea Fruit

Drupe 1.5–2.5 cm long, ovoid on obovoid-pyriform, orange-yellow at maturity The

hairless fruit has a diameter of 10 to 15 mm and glossy yellow at maturity. It is observed

that they have a bittersweet taste. Gmelina arborea is not considered a threat, and in many

countries, as well as in large numbers in plantations, it can be found growing in the wild.

(Zambesiaca and Fernandes, et al., 2005)

Uses of Gmelina arborea

 Gmelina arborea is cultivated as an ornamental, avenue and shade tree in urban and

peri-urban regions. It is also used in coffee and cocoa plantations to protect young crops

and to suppress harmful grass. It's useful as a firebreak because it suppresses undergrowth

and its leaves rot easily. It is often planted as a wind and a hedge. It has the potential for

reforestation in dry forest areas. Throughout tropical Asia, the roots of bark, leaves, fruits

and seeds are used in Hindu medicine. The fruit and bark have medicinal properties against

bilious fever. Leaf sap is used as a demulsifier to cure gonorrhea and cough, and is used for

wounds and ulcers. Roots are known to have tonic, nausea and laxative properties, and

flowers have been used to treat leprosy and blood disorders. The fruits are also edible. The

leaves are commonly used as cattle feed and in the cultivation of silkworms. Wood ash and

fruit contain very strong yellow colours. The flowers produce a large amount of nectar from

which a high-quality honey is produced. (Adam and Krampah et al., 2005)

Ecology of Gmelina arborea

Gmelina arborea is quite widespread in its natural distribution area, where it occurs

in habitats ranging from rainforests to drier deciduous forests. It reaches its maximum size

in the more humid forests of Myanmar, particularly in the wet fertile valleys. It can grow to

an altitude of up to 1400 m (e.g. in Ethiopia), but then it is typically stunted. This thrives in

temperatures with an average annual temperature of 21–28 ° C, with an average maximum

temperature of 24–35 ° C in the hottest month and a mean minimum temperature of 18–24

° C in the coldest month. Annual rainfall in its natural range varies from 750 mm to 4500

mm, but the optimum is round annual rainfall of 1800–2300 mm in areas with a dry period

of 3–5 months and a relative humidity of at least 40%. Although gmelina can be found on a

variety of soils, it prefers deep, moist soils with a large supply of nutrients. Growth in
leached acid soils is poor. Once set in poor conditions, trees frequently remain stunted and

become little more than a shrub. For plantations, well-drained fertile soil is required and

waterlogged sites are not maintained. Gmelina is an opportunistic species in the rainforest,

and has been listed as a long-lived explorer. It has a high demand for heat. It has become

common in many African countries, where it can be very invasive. (Adam and Krampah et

al., 2005)

Anatomy of Gmelina arborea

Gmelina arborea has been extensively established for commercial purposes in

Costa Rica. Such new phrases for Gmelina cause differences in anatomy in the secondary

xylene of bushes growing in the plantations. The purpose was once to determine the xylem

anatomy version prompted with the aid of the ecological conduction difference. Measured

fiber sizes, cross-section axial parenchyma, vessel parameters and light. The outcomes

confirmed that, given modifications in ecological situations, several anatomical traits

remained stable, particularly radial parenchyma and anatomical aspects less affected by

using altitude. On the other hand, the vessels axial parenchyma and fiber were less steady

due to longitude, latitude, elevation and precipitation. (Moya and Tomazello et al., 2008)

Morphology of Gmelina arborea

It is a moderately deciduous tree with a straight trunk and various spreading

branches, which make a large, shady crown with a white grey lenticel late bark, exfoliating

in thin flakes. It has a clear bole of 6.0-9.0 meters and a width of 1.5-2.5 meters. Branchlets

and young parts are covered in fine white, mealy pubescence. The leaves are plain,

opposite, broadly ovate, cordate, glandular, glabrous above when mature and filled-
tomentose beneath. It has reached its largest size in the mixed forests of the humid zone, as

in the eastern sub-Himalayan line, Assam, and elsewhere in southern India. (Gmelina

arborea, 2019)

Components of Gmelina arborea

Gmelina arborea's main chemical components includes lignans, glycoside iridoid,

flavonoids, flavones, glycoside flavones and sterols. This evaluation involves all the

sources and useful instructions for further study of usage G. arborea. (Arora and Tamrakar

et al., 2017)

Phytochemical Screening

It relates to the collection, screening and identification of plant-based medicinally

active substances. Flavonoids, alkaloids, carotenoids, tannins, vitamins and phenolic

compounds are some of the bioactive substances that can be obtained from plants.

(American Journal of Phytomedicine and Clinical Therapeutics, 2019)

Phytochemicals

These are secondary metabolites that contribute to taste and color (Craig, 2009).

Phytochemicals are bioactive substances that work with nutrients and dietary fiber to

protect from disease (Sangeeta and Sujata, 2006). These compounds are thought to be

responsible for much of the disease protection provided by diets rich in fruit, beans, peas,

cereals and plant-based beverages such as tea and wine. They can be categorized as

phenolic acids, flavonoids and stilbenes / lignans based on their chemical structure.

Flavonoids are further classified into anthocyanins, flavanes, flavanes, isoflavones and

flavonols, among others. (Arts and Hollman et al., 2005)

 Bioactive Compounds

Bioactive compounds are extra-nutritional elements that typically occur in small

amounts in foods. They are being researched intensively to determine their effects on

health. The driving force behind this scientific investigation was the result of a number of

epidemiological studies that confirmed the protective effects of plant-based diets on

cardiovascular disease (CVD) and cancer. A number of bioactive compounds have been

found. Such compounds vary widely in chemical structure and function and are grouped

together accordingly. Phenolic compounds, including subcategories, flavonoids, are found

in all plants and have been extensively studied in cereals, legumes, nuts, olive oil,

vegetables, berries, tea and red wine. Many phenolic compounds have antioxidant

properties, and some studies have demonstrated favorable effects on thrombosis and

tumorigenesis and promotion. (Etherton and Hecker et al., 2002)

Flavonoids

Flavonoids are a diverse group of phytonutrients present in almost all fruits and

vegetables (plant chemicals) that, together with carotenoids, are responsible for the vivid

colors of fruits and vegetables. Flavonoids are the largest group of phytonutrients with

more than 6,000 varieties. Qercetin and kaempferol are some of the best-known flavonoids.

Flavonoids are part of the phytonutrient polyphenol group. Polyphenols have traditionally

been used in Chinese and Ayurvedic medicine, in addition to antioxidant and anti-

inflammatory action, they are associated with skin defense, brain function, blood sugar and

blood pressure control. While flavonoids have strong antioxidant function, according to

World's Healthiest Foods, they occur in the bloodstream at a relatively low level compared
to antioxidants such as vitamin C and vitamin E. This can reduce their total antioxidant

strength, thus minimize their cancer-fighting effectiveness. (Szalay, 2015)

Chick Chorioallantoic Membrane Assay

Many laboratories investigating angiogenesis use a process in which the developing

chicken embryo is separated from the shell and transferred to the petri dish. The author's

lab uses intact eggs. Access to CAM is achieved by cutting a shell window. A sample may

be applied to the CAM, the membrane that surrounds the embryo, either directly as a solid

(for raw samples), implanted in a slow-release polymer pellet, or dried on a plastic cover.

The time of application, post fertilization, is more important than the period at which the

development of the blood vessels is registered. Many laboratories are trying to score

angiogenic reactions on some arbitrary scale, e.g. 0–4, but the findings are considered

either positive or negative. (Riordan, 2001)

Mallard Duck

The Mallard duck is a duck of medium size. Quite often it is a bit lighter than most

other dabbling ducks. For the most part, dabbling ducks are feeding on the surface instead

of swimming. The Mallard duck is 50 to 65 cm in length and has an 81 to 98 cm wing span.

The bill is 4.4-6.1 cm, the tarsus is 4.1-4.8 cm and the chord of the wing is 25.7-30.6 cm.

There is a shiny bottle-green head and white collar in the breeding Mallard drake. The head

is divided from the dark purple tinged breast by a white collar, a light gray neck and black

brown wings. The drakes have a black back on the dark tail with white tips. The drake's bill

is a black-tipped, yellowish orange. But the Mallard's female bill is typically darker, from

black to mottled orange. (Mallard Duck Characteristics & Breed Information, 2018)
 Hypotheses of the Study

Ha: There is a significant difference between the three different treatments of Gambhari

(Gmelina arborea) to the angiogenic inhibition using Mallard (Anas platyrhynchos) duck

embryo.

Ho: There is no significant difference between the three different treatments of Gambhari

(Gmelina arborea) to the angiogenic inhibition using Mallard (Anas platyrhynchos) duck

embryo.
 METHODOLOGY

Research Design

The investigation makes use of Completely Randomized Design (CRD) as an

experimental method by extracting and testing the effectiveness of the different treatments

of G. arborea to the A. platyrhynchos. Treatments were designated out randomly to the test

subjects for the examination configuration utilized. Five hundred (500) mg of the different

parts of the Gmelina arborea was mechanically pounded utilizing a mortar and pestle.

From that point forward, the pounded plant had experienced a decoction procedure. The

samples produced were controlled to the mallard duck embryo with an estimation of three

(3) cc utilizing a syringe. Likewise, to the next sample which is the commercial product and

the distilled water it was additionally estimated straightforwardly with a syringe with an

estimation of three (3) cc.

The different treatments were utilized in the experimentation are the following:

Negative Control: 0.5g Distilled Water

Positive Control: 0.5g of Commercial Product

Treatment 1: 0.5g of G. arborea leaves concoction

Treatment 2: 0.5g G. arborea fruit exocarp concoction

Treatment 3: 0.5g G. arborea roots concoction

 Entry Protocol

Location and Duration of the Study

The plant utilized will be collected at Sugarland Lumbo, Valencia City, Bukidnon.

The initial identification of the plant will be done in the Biology Laboratory of Central

Mindanao University and will be confirmed by a biology expert. The phytochemical

examination of Gmelina arborea leaf, fruit exocarp, and root extract in the Laboratory of

Central Mindanao University. The Mallard duck embryo will be bought at the Philippine

Institute of Traditional and Alternative Health Care (PITAHC) at Davao City. Moreover,

the utilization of the treatments and assessment of the blood vessel formation of the mallard

duck embryo and the decoction procedure will be done at the Biology Laboratory of

Valencia National High School, Valencia City, Bukidnon. The examination will be

done_____________.

Equipment and Materials Used

To fill in as the experimental subject, 15 mallard duck embryo were utilized in the

investigation. Fifteen (15) pieces of syringe were utilized in directing the experiment; in

every treatment there are three (3) cc concoctions infused to the mallard duck embryo. The

researchers also utilized three (3) sets of nitrile gloves for safety measures.

Experimental Subjects

Fifteen (15) mallard duck embryo of three (3) days old will be purchased from the

Philippine Institute of Traditional and Alternative Health Care (PITAHC) situated at Davao
City and will spontaneously place on the incubator for conservation. The test subjects were

divided into six (6) groups in accordance to their different treatments.

Animal Care and Safety

Collection and Identification of Plant Materials

Preparation of Plant Sample

Decoction of Plant Samples Method

Phtytochemical Analysis

The bioactive compounds of fruit exocarp, leaf and root of G. arborea will be

screened at Central Mindanao University Musuan, Dologon Bukidnon.

Classification of Treatment Groups

In the study, fifteen (15) mallard embryo were used as test subjects that were

purchased in the Philippine Institute of Traditional and Alternative Health Care (PITACH)

located at Davao City, Philippines. To utilize the research design chosen by the researchers

which is the Completely Randomized Structure (CRD), the test subjects were divided

randomly into five (5) treatment groups; Negative Control (Distilled Water), Positive

Control (0.5g of Commercial Product), Treatment 1 (0.5g of G. arborea leaves concoction),

Treatment 2 (0.5g of G. arborea fruit exocarp concoction), and Treatment 3 (0.5g of G.

arborea roots concoction), where every treatment groups had three (3) Mallard duck

embryo as replicates.
 Preparation of Treatments and Induction of Methrotrexate

Chick Chorioallantoic Membrane Assay

Duck Chorioallantoic Membrane Assay (CAM) Test The 3-day old fertilized duck embryo

were incubated for 7 days at 37.50Cand 70% humidity. Prior to windowing, a

HEALTHPRO gauze absorbed 70% Bandage Isopropyl alcohol was cleaned into the shell

of the ducks, A window in the egg shell about 1x1 cm was made to expose the CAM for

access to experimental control. The test plant extract was ingested on the sterile filter paper

discs. At that point, the treated filter paper discs were placed onto the CAM. The treated

eggs were sealed with clean plastic tape and were incubated for two days. On the tenth

(10th) day, the eggs were exposed for experimental treatments since the developing CAM

vasculature was prepared to sprout in response to additional pro-angiogenic improvements

and were very responsive to antiangiogenic factors. On the twelfth day of incubation, the

CAMs were gathered by clearing the hard shell leaving soft membrane covering the

embryo. The shell-less embryo was moved to a petri dish and 5 mL of the amniotic liquid

was removed utilizing a 10mL syringe with a needle. Duck embryo that were dead before it

was collected, that is one day after the soaked filter paper, were replaced and a similar

procedure was performed until they attained at the twelfth (12th) day of incubation as

described in the procedure of Fajardo et al., (2015)

Disposing of Mallard Duck Embryo Procedure

Data Gathering Procedure

Data Analysis

Photo Documentation
 Classification of Treatment Groups

In the study, fifteen (15) mallard embryo were used as test subjects that were

purchased in the Philippine Institute of Traditional and Alternative Health Care (PITACH)

located at Davao City, Philippines. To utilize the research design chosen by the researchers

which is the Completely Randomized Structure (CRD), the test subjects were divided

randomly into five (5) treatment groups; Negative Control (Distilled Water), Positive
Control (0.5g of Commercial Product), Treatment 1 (0.5g of G. arborea leaves concoction),

Treatment 2 (0.5g of G. arborea fruit exocarp concoction), and Treatment 3 (0.5g of G.

arborea roots concoction), where every treatment groups had three (3) Mallard duck

embryo as replicates..