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. (KIMAHANGLAN UG CITATION)

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 flower 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 flower 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 flower 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 reducing the blood vessel formation to the A.

platyrhynchos?

Objectives of the Study

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

arborea) fruit exocarp, leaf, and flower 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 flower.

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 reducing the blood vessel formation to

the A. platyrhynchos.

Significance of the Study

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

arborea) fruit exocarp, leaf, and flower 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 flower extract that can inhibit angiogenic signaling

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

specifically on its fruit exocarp, leaf, and flower 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 0arborea) 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 millimeter 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)

Alkaloids

Medicinal Properties of alkaloids has many differences. Alkaloids are highly

important active components in natural herbs wherein some compounds were already

profitably developed into chemotherapeutic drugs(Huang, Gao and Chen, 2008) and have a

wide conveyance in the plant realm and essentially exist in higher plants (Glencross, 2016).

Numerous alkaloids have local sedative properties, however clinically they are occasionally

utilized for this reason. Two alkaloids, vincristine and vinblastine (from Vinca rosea), are

broadly utilized as chemotherapeutic agents in the treatment on various kinds of types of

cancer. Much of the time, plant tissue is prepared to obtain arrangements of the alkaloids.

The alkaloids are then recuperated from the solution by a procedure called extraction,

which includes dissolving a few segments of the mixture with reagents. Various alkaloids
should then be isolated and purified from the mixture, then be isolated and purified from

the mixture. (Gaur, 2018).

Tannins

Tannins are broadly transmitted in plants and happens in particularly high amounts in

the bark of specific trees (e.g., oak) and in galls (Trugo and Baer, 2003) and are composite

polyphenols substances found in plants, especially in pulses, with the property to accelerate

proteins in aqueous medium (Walter and Piechulla, 2011). They are generally bounteous

and multipotent molecules showing antioxidant, proapoptotic and antiangiogenic activity

and in this manner being compelling in the treatment of degenerative diseases including

initation and progression of tumors. (Rekha, Aradhya and Jayashree, 2015)

Chick Chorioallantoic Membrane Assay

Many laboratories investigating angiogenesis use a process in which the developing

chicken or duck 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 Gambhari (G. arborea) to the Mallard (A. platyrhynchos) duck embryo. Treatments will

be designated out randomly to the test subjects for the examination configuration utilized.

Two hundred (200) grams of the different parts of the Gambhari (Gmelina arborea) will be

mechanically pounded utilizing a mortar and pestle. From that point forward, the pounded

plant had to undergo a decoction procedure. The samples produced will be 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 will be additionally

estimated straightforwardly with a syringe with an estimation of three (3) cc.

 The different treatments will be utilized in the experimentation are the following:

Negative Control: 5.0ml Distilled Water

Positive Control: 5.0ml of Commercial Product

Treatment 1: 5.0ml of G. arborea leaves concoction

Treatment 2: 5.0ml G. arborea fruit exocarp concoction

Treatment 3: 5.0ml G. arborea flowers concoction

Entry Protocol

A permit from Valencia National High School's school principal will be secured for

the researchers to be able to conduct their study outside of the premise of the school.

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 taxonomy specialist. The phytochemical

examination of Gambhari (Gmelina arborea) leaf, fruit exocarp, and flower extract will be

located in the Laboratory of Central Mindanao University. The Mallard (A. platyrhynchos)

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 (A. platyrhynchos) duck embryo procedure
will be done at the Biology Laboratory of Valencia National High School, Valencia City,

Bukidnon. The experimentation will be done on March to September 2020.

Equipment and Materials Used

Fifteen (15) Mallard (A. platyrhynchos) duck embryo, mortar and pestle, fifteen (15)

pieces of monoject thirty-five (35) ml catheter tip syringes, health pro gauze, amber,

electric blender, commercial product (methotrexate), distilled water, sterile filter paper

discs,filter paper, incubator, fifteen (15) vials, masking tape, scissors, weighing scale,

record book, petri dish, three (3) pairs of nitrile gloves, and a beaker.

To fill in as the experimental subject, fifteen (15) Mallard (A. platyrhynchos) duck

embryo will be utilized in the investigation. Fifteen (15) pieces of monoject thirty-five (35)

ml catheter tip syringes will be utilized in directing the experiment; in every treatment there

are three (3.0) cc concoctions infused to the mallard duck embryo. The researchers will also

utilize three (3) sets of nitrile gloves for safety measures.

Experimental Subjects

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

be divided into five (5) groups in accordance to their different treatments.

Research Animals

Fifteen (15) three-day fertilized duck embryos will be obtained from the Philippine

Institute of Traditional and Alternative Health Care (PITAHC) situated at Davao City. Egg

viability will be determined using the candle method for any sign of embryo formation
assisted by a licensed veterinarian. The eggs will be randomly grouped and labeled

according to treatment. The eggs will be placed in the incubator at a constant temperature

of 37.50 ℃ and at a constant humidity.

Collection and Identification of Plant Materials

The chosen pieces of the Gambhari (G. arborea) plant to be specific; leaves, fruit

exocarp, and flowers will be gathered at the vicinity of Valencia National High School. The

gathered Gambahri (G. arborea) parts will be set inside on a container and will be washed

completely with refined water to expel any dirt and exceptionable microorganisms. The

preliminary identification of the plant will be done in the Biology Laboratory of Central

Mindanao University and will be confirmed by a taxonomy specialist.

Preparation of Plant Sample

The collected plant will be thoroughly washed with water from the tap to remove all

the impurities that accumulated on the soil and then air dried at room temperature for seven

(7) days, which will be mixed regularly to prevent mold. The different dried parts of the

Gambhari (G. arborea) plant will be then gutted into pieces and then mechanically

pulverized using an electric blender for about two hundred (200) grams.

Decoction of Plant Samples Method

In the preparation of the decoction procedure of the plant, five (5.0) grams of

powdered fruit exocarp, leaves and flowers of Gambhari (G. arborea) plant will be

independently added to twenty five (25) ml of water. It will be heated for five (5) minutes

in a sterilizer to ensure that the plant is germ free and will be then boiled in an amber for

thirty (30) minutes to produce a concoction, from that point onward, the concoction will be
left at a room temperature for around ten (10) minutes as depicted in the procedure of

Ucang J., (2018). From that point onward, the three (3) different treatments of Gambhari

(G. arborea) with the measurement of three (5.0) ml and will be set in the three (3)

sterilized vials provided by the researchers. The decoction procedure will be done at the

Biology Laboratory of Valencia National High School.

Phtytochemical Analysis

The bioactive compounds of fruit exocarp, leaf and flower of Gambahri (G.

arborea) will be screened at Central Mindanao University Musuan, Maramag Bukidnon.

Phytochemical analysis will be conducted in Natural Products Research and Development

Center (NPRDC). The results will be examined based on the compound responses showed

by the addition of various test solutions and with few methods applied. These procedures

are as follows:

1. Test for Alkaloids

a. Dragendroff’s Test

Filtrates treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide).

Use three (3) ml of plant sample and add one (1) ml of the solution of Potassium Bismuth

Iodide of red precipitate indicates the presence of alkaloids.

2. Test for Tannins

a. Ferric Chloride Test

Candle Method
 Classification of Treatment Groups

In the study, fifteen (15) Mallard (A. platyrhynchos) duck embryo will be used as

test subjects that will be purchased in the Philippine Institute of Traditional and Alternative

Health Care (PITAHC) located at Davao City, Philippines. To utilize the research design

chosen by the researchers which is the Completely Randomized Design (CRD), the test

subjects will be divided randomly into five (5) treatment groups; Negative Control (5.0ml

Distilled Water), Positive Control (5.0ml of Commercial Product), Treatment 1 (5.0ml of

G. arborea leaves concoction), Treatment 2 (5.0ml of G. arborea fruit exocarp concoction),

and Treatment 3 (5.0ml of G. arborea flower concoction), where every treatment groups

had three (3) Mallard (A. platyrhynchos) duck embryo as replicates and will be infused

using a monoject thirty-five (35) ml catheter tip syringe with the measurement of three

(3.0) cc.

Duck Chorioallantoic Membrane Assay

The 3-day old fertilized duck embryo will be incubated for 7 days at 37.50℃ and

70% humidity. Prior to windowing, a HEALTHPRO gauze absorbed 70% Bandage

Isopropyl alcohol will be cleaned into the shell of the ducks, a window in the egg shell

about 1x1 cm will be made to expose the CAM for access to experimental control. The test

plant extract will be ingested on the sterile filter paper discs. At that point, the treated filter

paper discs will be placed onto the CAM. The treated eggs will be sealed with clean plastic

tape and will be incubated for two days. On the tenth (10 th) day, the eggs will be exposed

for five (5) experimental treatments; Negative Control (3.0 cc Distilled Water), Positive

Control (3.0 cc of Commercial Product), Treatment 1 (3.0 cc of G. arborea leaves

concoction), Treatment 2 (3.0 cc of G. arborea fruit exocarp concoction), and Treatment 3

(3.0 cc of G. arborea flowers concoction), since the developing CAM vasculature will

prepare to sprout in response to additional pro-angiogenic improvements and are very

responsive to antiangiogenic factors. On the twelfth (12th) day of incubation, the CAMs will

be gathered by clearing the hard shell leaving soft membrane covering the embryo. Aisha A

et al., (2009). The shell-less embryo will be moved to a petri dish and 5 mL of the amniotic

liquid will be removed utilizing a monoject thirty-five (35) ml catheter tip syringe. Then,

the blood-vessel formed will be manually counted with the assistance of a licensed

veterinarian.

Disposing of Mallard Duck Embryo Procedure

After the experiment, the Mallard duck embryos will be placed in an ecological bag

and blocked for five (5) hours at a temperature of 212 ° C, so as not to contaminate. The

embryos will be sealed and will be properly buried in a composting pit.

Data Gathering Procedure

In getting the number of blood vessel formed on the five (5) treatments of the

Mallard (A. platyrhynchos) duck embryo, the researchers will manually count the blood-

vessel formation with the assistance of the authorized veterinarian. Then again, as far as

having a blood vessel of the Mallard (A. platyrhynchos) duck embryo before and after the

exposure of the different treatments of the concoctions from the Gambhari (G. arborea)

plant and the commercial product, the researchers will utilize a similar strategy to decide if

the blood vessel formation had any kind of effect after infusing the concoctions from the

various parts of the Gambhari (G. arborea) and the commercial product, methotrexate. On

the tenth (10th) day of incubation, the researchers will equally infuse the Mallard (A.

platyrhynchos) duck embryo with a three (3.0) cc using monoject thirty-five (35) ml
catheter tip syringe in every treatment with three (3) replicas. After placing the treatments,

it will be putted back on the incubation prior on processing the blood-vessel formation on

the mallard duck embryo for two (2) days. On the twelfth (12th) day the CAMs will be

assembled by clearing the hard shell leaving soft membrane covering the mallard duck

embryo and will be placed on a petri dish for manually counting the blood-vessel formed.

Data Analysis

The Analysis of Variance (ANOVA) will be utilized to evaluate and compare the

data that will be accumulated in the study. It determines whether the concoctions from the

various parts of Gambhari (G. arborea) plant significantly affected on reducing the number

of blood vessel formation of the Mallard (A. platyrhynchos) duck embryo. Likewise, it will

be utilized to determine any significant effect between the three (3) concoctions from

Gambhari (G. arborea) plant and to compare the efficacy of the most effective concoction

from the plant to the commercial product after two (2) days of being tested by the

treatments. The analysis of data will be completed utilizing SPSS application provided by

the researchers. In addition, the researchers will be guided by a statistician to gather

accurate results.

Photo Documentation

In conducting the study, photographs will be taken for documentation utilizing an

android cell phone. Every photograph will be taken by the researchers and serve as a proof

along the direct of the examination.