VIRGEN MILAGROSA UNIVERSITY FOUNDATION

Martin P. Posadas Avenue, San Carlos City Pangasinan

COLLEGE OF MEDICINE

IN-VITRO MITOTIC 1

IN-VITRO MITOTIC ACTIVITY of JOB’S TEARS (COIX LACRYMA-JOBI)

Krizza Katrina Mae D. Antiporda, Kevin George D. Calugay, Serasingha Pathiranage Dumindu

Gajanayaka, Shiela Mae M. Gutierrez, Abrik Manandhar, Shrijana Pakhrien, Hanna Marie D.

Penaverde, Kayle Paolo S. Pinlac, Prabin Silwal

ABSTRACT

The antimitotic property of Job’s tears seed extract was determined by using onion (Alium cepa).

Job’s tears seed was subjected into phytocytochemical testing for its components. The roots of

the onion were used and were placed into different concentrations of the Job’s tears extract,

together with a positive and negative control. They were measured and analyzed macroscopically

in the seventh day and in the 14th day. After 14 days, the roots were processed for microscopic

examination. The different phases of mitosis were counted and computed by getting the mitotic

index (MI= number of dividing cell counted divided by 1000). The study used ANOVA method

for the interpretation of the results.

Based on the result of the computed Mitotic Index, Job’s tears extract proved its anti-mitotic

activity. The results showed that with an increased concentration of the Job’s tears extract the

mitotic activity in the root tip of the onion was decreased. 100% concentration of the extract
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IN-VITRO MITOTIC 2
showed much of its anti-mitotic property among the other concentrations. The result of the

present study will provide a new perspective and wider horizon in utilizing the seed extract of the

Job’s tears in medical field. Furthermore, the outcome of this study may serve as fundamental

reference on the development of this plant anti-mitotic property that will aid for the natural

process of treating of cancer.

Keywords: Mitotic Index (MI), Analysis of Variance (ANOVA), Onion (Alium cepa), Job’s

Tears (Coix lacryma-jobi)

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IN-VITRO MITOTIC 3
Chapter 1 of 75. And there are 59,000 people dying

from cancer annually. 13 out of 100 males

INTRODUCTION
and 14 out of 100 females in the Philippines
Throughout the world, cancer is
would have had some form of cancer if they
considered one of the leading causes of
would have lived up to age 75. Eleven out of
mortality and morbidity, having an
100 males and 7 out of 100 females would
approximately 14 million new cases and 8.2
have died from cancer before age 75.
million cancer related deaths in 2012.
(Laudico, 2015)
According to the American Cancer Society,
Chemotherapy is used to treat cancer
one in eight deaths is due to cancer and it
and ease or lessen the cancer symptoms.
causes more deaths than tuberculosis, AIDS
Anti- cancer drugs act by the inhibition of
and malaria combined (American Cancer
DNA synthesis or by other process of the
Society, 2015).
cell’s growth cycle. Having to affect rapidly
In the Philippines, it was studied in
dividing cells, other non-cancerous cells will
2010 that cancer ranked third in leading
also be affected that harms the patients
causes of death in the country. Wherein 75%
through its side effects and adverse
of cancer patients are 50 years old and above
reactions. This phenomenon led to many
while 3.2% belong to the pediatric age
studies and researches on the discovery of
bracket. An estimate from Philippine Cancer
new chemotherapeutic drugs that can lessen
resources in 2012, 98,200 people are newly
or can even omit side effects from the
diagnosed with cancer per year. There are
treatment. Scientists and drug formulators
14.8% risks of getting cancer before the age
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IN-VITRO MITOTIC 4
focus on developing new anticancer drugs used for adornment and decoration.

from organic or natural origins and plant- Ornamental beads were produced out of the

derived drugs to achieve less side effects various seed colors (e.g., yellow, white,

and moreover to provide cheaper and purple). The species name of the

affordable anticancer medication for wary aforementioned plant is Coix lacryma-jobi,

cancer patients (American Cancer Society, and commonly known as “Coïx”, “Larmes

2015). de Job”, or “Adlay”. A notable description

of the Job’s tears is its robust broad-leaved

Furthermore, the usage of medicinal
loose-growing branched grass up to 1.5 m
plants has provided significant information
high, and its bead is described by Armstrong
and development in different health
(1999), as a “very hard, hollow structure
institutions altogether, which is followed by
(called an involucre) containing a minute
the introduction and conception of new,
fertile female flower and two sterile flowers.
naturally-derived drugs. Consequently,
Pollen-bearing male flowers are produced
people were able to learn the genetic and
on a slender stalk that extends out of the
biochemical basis of disease and compounds
bead through a tiny pore. Two feathery
which have brought innovation and
stigmas from the fertile female flower also
opportunities in the field of medicine.
protrude from the pore—ready” (p.1). Also,
Hence, this research aimed to
Job’s Tears is used as food given its
understand the anti-mitotic property of Job's
indigenous and abundant nature in tropical
tears (Coix lacryma-jobi), a medicinal plant
Asia, and as extensively homegrown at the
discovered in China which is prevalently
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IN-VITRO MITOTIC 5
edge of rice fields in Java and Celebes are assumed to cause cancer. (International

(Armstrong, 1999). Agency for Research on Cancer, 2012) Cell

division is the feeding mechanism of cancer

The accelerated and uncontrollable
to create abnormal growth and cause the
body cells’ division and dispersion over
death of cells. Gradually, the normal tissues
other organs are characterized in all cases of
losses control over the cancer cells. These
cancer. Cancer can begin at any part of the
cells gain independence for their own
human body as composed by trillions of
growth until it is able to program cell death
cells, and consequently halts the orderly
throughout other parts of the body (Nature
course of cell cycle in which old or damaged
Education, 2014).
cells are being replaced by new ones

(National Cancer Institute, 2015). This Cell division is required by the

malignancy prompts the formation of tumors human body to generate new cells. The

as elaborated by National Cancer Institute human, being a diploid organism, has 23

(2015), “as cells become more and more homologous pairs of chromosomes. These

abnormal, old or damaged cells survive chromosomes are split into two equal sets

when they should die, and new cells form when the DNA of the nucleus of the cell is

when they are not needed. These extra cells divided during the course of mitosis in the

can divide without stopping and may form cell cycle. The one cell (mother) fragmented

growths called tumors” (p. 1). into two (daughter) explains the genetic

Furthermore, genetic susceptibility resemblance of all cells in the body (Khan

and environmental toxins such as benzene Academy, n.d.)

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IN-VITRO MITOTIC 6
The life of a cell undergoes a process chromosomes, likewise, prepares for the last

called cell cycle which systematically phase of interphase. Lastly, the G2 phase

consists of its creation from the preceding which gives the finale for the cell to

cell cycle division up until its own division continuously grow before undergoing

in order to produce two new cells. One division into two independent cells. Aside

complete series/cycle of cell cycle entails from these succeeding phases, the G0 phase

two major phases, named as interphase and exists outside the cell cycle, elaborated by

mitotic phase (OpenStax College, 2013). OpenStax College (2013) as “a resting phase

These two phases indicate the onset of of the cell cycle. Cells that have temporarily

cellular growth in the former while cellular stopped dividing and are resting (a common

division in the latter, then followed by the condition) and cells that have permanently

process of cytokinesis. ceased dividing (like nerve cells) are said to

First, the interphase which is be in G0.” (p. 114), hence an inactive state.

primarily segmented into three sequential

The Mitosis and Cytokinesis are
stages with decreasing duration as they
called as the mitotic phase it is the cell that
progress. The first stage of interphase is
takes between 1 and 2 hour during duration
termed as the G1 phase, in which cellular
of the phase. This phase cell was undergoes
growth takes place with retention of its
in to two major processes. The first one is
metabolic activity, thus ensuring the next
mitosis, mitosis completes, the contents of
stage meant for synthesis. S Phase proceeds
the nucleus. The nucleus was pulled apart to
after the G1 phase, wherein DNA replication
each other and its distributed in to two
occurs resulting to a twofold number of
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IN-VITRO MITOTIC 7
halves. Cytokinesis occurs, and dividing the attach, when the identical partner was

cytoplasm and the cell body into two new already attach the forming of the familiar X-

cells. The mitosis is divided into four major shape of sister chromatids will disappears

stages that take the place after interphase and this early phase, The nucleolus and the

and the next is prophase, metaphase, nuclear envelope also disintegrates. Near the

anaphase, and telophase. The process is end of prophase there may be an invasion of

followed by cytokinesis. The one type of a the nuclear location by microtubules from

cell division is mitosis that can result in two the mitotic spindle. The nuclear membrane

daughter cells. Each of them has the same has disintegrated, and the microtubules

number and kind of chromosomes as the connect themselves to the centromeres that

parent nucleus, typical of ordinary tissue adjoin pairs of sister chromatids. The

growth. kinetochore is a protein shape at the

centromere that is the point of attachment

Phases of Mitosis among the mitotic spindle and the sister

There are four basic phases of chromatids. This stage is referred to as late

mitosis these are prophase, metaphase, prophase or prometaphase to indicate the

anaphase, and telophase. The first phase is transition between prophase and metaphase.

Prophase, this stage was the loosely packed Metaphase is the second stage of

chromatin coils and condenses to make mitosis. Throughout this stage, the sister

visible chromosomes. Because during chromatids, with their connected

prophase stage, the chromosome becomes microtubules, line up along a linear pathway

visible and that’s why their identical partner within the center of the cell. A metaphase
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IN-VITRO MITOTIC 8
plate then forms in between the centrosomes formed nuclei that surround the genetic

which can be seen on the ends of the cell. material, uncoils in a way that the

The microtubules work to pull apart the chromosomes return to its loosely packed

sister chromatids and bring one from each chromatin. The nucleoli then reappear, and

pair to each side of the cell. then the mitotic spindle breaks and each new

cell receive its own complement of DNA,

The third stage is the anaphase. This
organelles, membranes, and centrioles. In
particular stage takes place over a few
this specific part of telophase, the beginning
minutes, it starts when pairs of sister
of the cytokinesis also starts the splitting of
chromatids are separated from one another,
the cell into halves.
which then forms individual chromosomes.

As the microtubules shorten, these Forms around the midline of the cell
chromosomes are pulled to opposite ends of during cytokinesis is a contractile band
the cell through their kinetochores. Every which is made up of microfilaments, this
end of the cell receives one partner from band namely, the cleavage furrow, squeezes
every pair of sister chromatids, making sure the two cells apart until they separate. New
that the two new daughter cells will contain cells are now formed and one of these cells,
same genetic material. the stem mobile, enters its own cell cycle;

capable of growing and dividing again while

The formation of two new daughter
the other cell transforms into a functional
nuclei at the ends of the dividing cell
cell of the tissue, typically replacing another
indicates the beginning of the final stage of
cell.
mitosis, which is the telophase. These newly
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IN-VITRO MITOTIC 9
Furthermore, not all cells that impact cell cycle regulation”. Proper

undergo mitosis also undergo cytokinesis regulation of the cell cycle therefore plays a

which is the finals step in cell division. major role and importance to human health

According to Normand & King (2010), “as a because cancer usually arises from the

highly regulated, complex process, it is not perturbations of cell-cycle regulation.

surprising that cytokinesis can sometimes

fail. Cytokinesis failure leads to both

OBJECTIVES
centrosome amplification and production of
Some researchers studied Job’s tears
tetraploid cells, which may set the stage for
in cancer and leukemia and said to be has a
the development of tumor cells”.
promising results. Job’s tears as Anti-cancer,

Schafer (1998) mentioned that, “the which have a property of inhibiting the

cell cycle is a complex process that involves cancer cell to grow and to divide more. The

numerous regulatory proteins that direct the object of this study is to observe the Anti-

cell through a specific sequence of events mitotic activity of Job’s tears in root tip of

culminating in mitosis and the production of onion under the microscope. This study aims

two daughter cells. Furthermore, this cycle to identify what phase of the mitosis thus the

can be altered to the advantage of many viral Job’s tears inhibits the most. To differentiate

agents and is often dysregulated due to the result using different concentration of

alterations either in oncogenes that the Job’s tears. The clinical significance of

indirectly affect the cell cycle or in tumor the different concentration of the extract.

suppressor genes or oncogenes that directly

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IN-VITRO MITOTIC 10
What its specific component of Job’s tears develop cancer (Ludington & Diehl, 2006).

responsible for its Anti-mitotic activity.

There are five standard methods of

treatment for cancer. Namely, they are

surgery, chemotherapy, radiation therapy,

THEORETICAL FRAMEWORK
immunotherapy and biologic therapy.

Cancer continues to be one of the Surgery can be used to prevent, treat, stage

leading causes of global mortality. Due to and diagnose cancer. Chemotherapy is a

the widely proven adverse effects of kind of cancer treatment that uses drugs to

majority of anticancer medications, recent eliminate cancer cells. Unlike surgery,

studies have extensively geared toward chemotherapy affects the entire body, not

looking on medicinal plants that have just a specific part. It works by targeting

biochemical components with potential rapidly multiplying cancer cells. Radiation

therapeutic value for cancer treatment. therapy uses certain types of energy to

shrink tumors or eliminate cancer cells. It

Research shows that certain risk
works by damaging a cancer cell’s DNA,
factors such as growing older, smoking,
making it unable to multiply. Biologic
sunlight, ionizing radiation, certain
therapy is a term for drugs that target
chemicals and other substance, some viruses
characteristics of cancerous tumors. Some
and bacteria, certain hormones, family
types of targeted therapies work by blocking
history of cancer, alcohol, poor diet, lack of
the biological processes of tumors that allow
physical activity or being overweight
tumors to thrive and grow (Fayed, 2009).
increase the chance that a person will
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IN-VITRO MITOTIC 11
According to the National Institute initiate this disease.

of Health stated 20-30 different theories on

The theory then implies that
aging in which according to them, “cancer is
antioxidant compounds will be beneficial in
a disease of aging”, which means that as the
reducing the formation or counteracting the
average lifespan of species increases, the
destructive effects of free radicals from
occurrence of the disease is visible. This
oxidizing sensitive biological molecules.
theory of the National Institute of Health
Scientific evidence also suggests that
associated with Denham Harman’s theory of
large doses of these antioxidants may
free radicals, stating that with accumulated
prevent chronic illness such as heart disease
free radicals may damage and oxidative
and cancer because these beneficial
stress, biochemical and cellular processes
compounds are thought to mop up unstable
begin to do more “incorrect” things a begins
chemicals in the body (free radical) that can
to progress. Also, Harman had invoked that
damage tissues and contribute to disease.
free radicals are causes of degenerative
Primary sources of naturally occurring anti-
diseases, such as cancer. Harman also
oxidants are whole grains, fruits and
argued that free radicals from oxygen is
vegetables. Plant-sourced food antioxidants
produced during normal respiration would
like vitamin C, vitamin E, carotenes,
cause cumulative damage which would
phenolic acid, and phytoestrogens have been
eventually lead to organismal loss of
recognized as having the potential to reduce
functionality, and ultimately death. These
disease risk. Moreover, a number of clinical
free radicals may oxidize nucleic acids,
studies suggest that the antioxidants in fruits
proteins, lipids or DNA and therefore can
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IN-VITRO MITOTIC 12
and vegetables, tea and red wine are the damaging effects of free radicals (Redmod,

main factors for the observed efficacy of 2007). Vitamin C and vitamin E helps to

these foods in reducing the incidence of quench the free radicals. While in the past

chronic diseases including heart disease and studies it was demonstrated and proven that

some cancers (Miller & Rigelhof, 2000). the antioxidant and vitamin E may also help

our body to prevent heart diseases. In fact,

Commonly sources of naturally of
the combination of vitamin C and E can help
anti-oxidants are whole grains, fruits and
to slow progression of cardiovascular
vegetables. Plant-sourced food antioxidants
diseases. According to Harvard Medical
came from vitamin C, vitamin E, carotenes,
School reported in 2002 that vitamin E plays
phenolic acid, and the phytoestrogens are
a role in helping people live longer, in
recognized and proven potential to reduce
strengthening the immune system (Hendler,
disease risk. Studies about the
Simon & Shuster, 2002).
antioxidants came from fruits and

vegetables, tea and red wine are the factors Application of Solvent Extraction

that have been observed in reducing Techniques is commonly used in different

incidence of chronic diseases including heart chemical, radiochemical, biochemical, and

disease and cancers (Miller & Rigelhof, pharmaceutical industries. According to the

2000). researches the studies was utilized the seed

parts to obtaining the extract solvent that

Vitamin C or also known as ascorbic
used in their respective studies. And
acid was proven as antioxidants that
researchers used different techniques in
combines and neutralizes the tissue-
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IN-VITRO MITOTIC 13
extracting the solvent depending on the to College of Pharmacy Laboratory for

plants’ and their natural characteristics, both Phytocytochemical test and Extraction.

chemically and biochemically. Some plants The onion is obtained from San

that they used are heated because the plants Carlos local market. Six onions are

cannot be exposed to a higher temperature prepared, the first four are placed in

like for example boiling because it will different concentration of Job’s tears 25%,

destroy the active component of the plant, 50%, 75% and 100% and labelled as A1,

thus the obtained results can lead to false A2, A3, and A4 respectively. The 5th onion

readings. is placed in distilled water which serves as a

negative control and labelled as B. The 6th

CONCEPTUAL FRAMEWORK
onion is placed in a solution with vitamin C
Job’s tears is collected from San
and labeled as C. The onion is kept for 14
Carlos, Pangasinan and authenticated in San
days to allow the growth of its roots. The
Andres Street, Malate, Manila City, Metro
roots are processed then for microscopic
Manila. After the authentication the seed
Examination.
from the plant was collected and was

crushed. The crushed seed sample is brought

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IN-VITRO MITOTIC 14

STATEMENT OF THE PROBLEM

This research aims to determine and

THRUPUT
1.Plant Authentication
2. Phytochemical analysis OUTPUT analyze the anti-mitotic Job tear’s extracts.
INPUT 3.In-vitro mitotic Job tear's
Job Tear's inhibition of jobs tears
seed extract 1. What are the active constituent of
- 25%
seed effective as
- 50% antimitotic. Job tear’s.
-75%
-100% 2. What is the mitotic activity of job'

tear using onion root as to the

following concentration

- 25%

- 50%

- 75%
Figure 1: Paradigm of the Study
- 100%

3. Is there significant different on the

following concentration to the

positive and negative control

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IN-VITRO MITOTIC 15
NULL HYPOTHESIS conducted at VMUF Research Center.

There is no significant inhibition Job’s

tears’ (Coix lacryma-jobi) seed extracts on SIGNIFICANCE OF THE STUDY

the Alium cepa roots.

The emergence of new synthetic
This was tested at 0.05 level of significance.
drug and the continuous increase in the

SCOPE AND importation of raw materials lead us to

DELIMITATION study the value of medicinal plants in

OF THE STUDY order to lighten the burden of the public

consumers with the extensive study

The study was conducted to
being made; it is possible that medicinal
determine the anti-mitotic of Job’s tears’
plants may become an alternative means
(Coix lacryma-jobi). The experimental
for commercially prepared medicines.
method that will be used is Clastogenecity

Bioassay eliciting data for analysis served As of today, cancer is the leading

as the basis in finding some proofs in order cause of death of some countries in the

to validate the written facts suggesting its world. Medical sciences continue to strive

anti-mitotic activity. towards cancer research and drug

development for the treatment of cancer.

Biological laboratory
However, the overall death rates for most
experimentation is conducted at VMUF
cancer cases continue to rise despite the
College of Pharmacy Laboratory and
modern technologies and innovations that
determination of the inhibition of root
have been made.
growth through microscopic analysis is
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IN-VITRO MITOTIC 16
The researchers find this study Medicine and Allied Medical

deemed to be significant in providing Professionals: The vital information

important evidence based findings regarding gathered will open more doors that aim to

the potential anti-mitotic property of Job’s promote knowledge and awareness for the

tears (Coix lacryma-jobi) in the following medical students and healthcare

clusters: professionals. It also interests to widen the

The Country: This study will provide a familiarity of the students towards the anti-

new perspective and wider horizon in mitotic cpotential of Job’s tears seed extract

utilizing Job’s tears (Coix lacryma-jobi) in against human cancer lines.

medical field. The University: This study may have

The Pharmaceutical Industry: The spearhead contribution to the improvement

outcome of this study may serve as of scientific community inside the university

fundamental reference on the development but also aims to be the forefront in

of this plant anti-mitotic property that will innovation outside the institution.

aid for the natural process of treating of

cancer.
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IN-VITRO MITOTIC 17
Chapter 2 effects of the solutions of varying

METHODOLOGY concentrations of Job’s tears’ (Coix

lacryma-jobi) on the anti-mitotic were

This presents the research design,
analyzed.
preparation of materials and set-ups that

includes the collection and preparation of The four solutions of varying

plant material, extraction present in the job concentrations of job tear’s extracts; 25%,

tear’s as well as formulation and 50%, 75% and 100% were designated as

preparation of the culture media utilizing four treatments of this study. Each

natural crude drugs and the statistical treatment was replicated two times.

treatment of data used in this study. Treatment with 0% level was designated as

the negative control treatment of the study

RESEARCH DESIGN and positive control with vitamin C tablets.

The study utilized experimental Clastogenecity bioassay were

method of research to determine and employed to evaluate the Anti-mitotic

analyze the anti-mitotic of job tear’s activity of the different concentrations of

extract. Experimental method of research Job tear’s extract using bulbs - Allium cepa,

aims to establish cause-and-effect family Solanaceae as test subjects.

relationships between variables (Pagoso,

2004). LOCALE OF THE STUDY

The Job’s tears’ (Coix lacryma-jobi)

The study made use of Complete
is collected from San Carlos, Pangasinan.
Randomized Design (CRD) where the
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The experiment will be conducted to VMUF the Job’s tears’ (Coix lacryma-jobi) whole

college of Pharmacy laboratory. plant will be cleaned and air dried at room

Data Gathering Procedures temperature for at least one week. The plant

Collection and identification of plant will then be authenticated by the San Andres

material: Street, Malate, Manila City, and Metro

Plant of Job’s tears’ (Coix lacryma- Manila.

jobi) is collected at San Carlos, Pangasinan; MATERIAL

Laboratory Apparatus

Electron microscope Test tube

Glass slide Test tube rack

Cover slip Stirring rods

Alium cepa bulbs Graduated cylinder

Erlenmeyer flask Pipette

Mortar and pestle Weighing scale

Crucible tong Reagent bottles

Beaker Petri dish

Water bath Glass slide and cover slip

Test Reagents

1% hydrochloric acid Bouchard’s reagent

Ethanol Concentrated sulfuric acid

Mayer’s reagent Kedde reagent

Wagner’s reagent Sodium hydroxide

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Glacial acetic acid Benzene

Gugo extract Hydrogen peroxide

Sodium chloride Ammonia

1% gelatin solution Acetocarmine

Petroleum ether Glycerin

0.5 N potassium hydroxide Test Materials

Vitamin C Distilled water

Job’s tears seeds Onion bulbs

by warming on steam bath for 1 or 2

min. Cool, filter, and then adjust the

PHYTOCHEMICAL SCRENING volume of the filtrate to 7ml by

PROCEDURE washing the residue on filter paper

A. Screening for Alkaloids with a sufficient quantity of 1%

Evaporate 70ml of the 95% hydrochloric acid. Add few grains of

ethanoic extract to dryness on a powdered sodium chloride to the

steam bath. Dissolve the residue in filtrate, shake and then refilter.

7ml of 1% hydrochloric acid, aided

Place 1ml of the filtrate into each of 4 second, 3 drops of Mayer’s reagent

small test tubes. To the first test tube, add 3 (Mercury iodide TS). In the third, 3 drops of

drops of Modified Mayer’s reagent Wagner’s reagent (Iodide and Potassium).

(Mercury Potassium Iodide TS). In the Finally in the fourth, 3 drops of Bouchard’s
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reagent (2% Iodide and 4% Potassium essentially dehydration reactions and

Iodide). therefore moisture must be excluded

B. Screening for Unsaturated Sterols and in each of the experimental steps.

Triterpenes B1. Liebermann-

Evaporate 30ml of the 95% Bourchardt’s Test – to 5ml of the

ethanoic extract to dryness on a filtrate in a suitably dry test tube, add

water bath. Cool the residue to room 0.3ml of acetic anhydride and mix

temperature and add 15ml of light gently. Add one drop of concentrated

petroleum ether. Mix well and filter. sulfuric acid. Observe any color

Repeat with additional volumes of change immediately and every 5

petroleum ether until colorless. mins thereafter over 50 mins. period.

Combine the ethereal filtrates, set Run this test concurrently with 5ml

aside the defatted residue for portions of standard solutions

screening for flavonoids and prepared from plants known to

leucoanthocyanins. contain unsaturated sterols or

Evaporate the combined triterpenes.

ethereal filtrates to dryness and then B2. Salkowski Test – transfer 5ml of

dissolve the residue in 15ml of the filtrate to a dry test tube and

chloroform. Add a pinch of perform a ring test with concentrated

anhydrous sodium sulphate to the sulfuric acid. Shake for 1 to 2

filtrate and divide equally into three minutes and note the color change.

dry test tubes. The following test are

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IN-VITRO MITOTIC 21
B3. Color control – add 5ml of the temperature for 1 hour before

filtrate to the third test tube. Add no recording the result as negative.

reagents. This tube is to serve as a To Test tube #2, add 0.5ml of

color control for both test. concentrated hydrochloric acid and

3-4 magnesium turnings. Observe

C. Screening for Flavonoids carefully for a color change (to

Dissolve the defatted residue green, red, etc.) within 10 minutes

from section B-3 in 30ml of 50% which is indicative of the presence

ethanol filtrate and place 1-2ml of of flavanols. If a definite color is

the filtrate in each of the three test formed, cool and dilute with an

tubes. equal volume of water and add

To Test tube #1, add 0.5ml of 1.0ml of octyl alcohol. Shake and

concentrated hydrochloric acid and allow to separate. The color in the

warm in a steam bath for about a octyl alcohol layer is due to

minute and observe the color change. aglycones while the color in the

The development of a red-violet aqueous layer is due to glycosides.

color is indicative of the presence of

leucoanthocyanidins. Color

formation may be slow. If the color

is not immediately apparent, allow

the test solution to stand at room D. Screening for Steroid (Cardio active)

Glycosides
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IN-VITRO MITOTIC 22
D1. Presence of Unsaturated Sterols extract in an evaporating dish and

(Liebermann-Burchard Test) – use dry on a steam bath. Add 3mL of the

the result of section B1 ferric chloride reagent (mix 0.3ml of

D2. Presence of Unsaturated 10% ferric chloride solution with

Lactones – since the following three 50mL of glacial acetic acid), stir to

test involve color reactions, it is mix well and transfer to a small test

necessary to run concurrent test with tube. With the test tube held at 45⁰

the control sample. angle, layer 1ml of concentrated

D2a. Kedde Reaction. To 5ml sulfuric acid by allowing it to flow

of the 80% ethanoic extract in an down the inside wall of the test tube.

evaporating dish. Add 5ml of Kedde Avoid shaking or agitating the test

reagent (2g of 3,5-dinitrobenzoic tube at this point. Observe for a

acid in 100ml of ethanol) and mix purple ring at the interface which

well with a glass stirring rod. To the would indicate the presence of 2-

mixture, add 2ml of 1N sodium deoxysugars.

hydroxide. Mix and observe color

development. A purple color is E. Screening of Saponins

positive indication of the presence of E1. Froth Test

the unsaturated lactone ring. Take a volume of the

D2b. Presence of 2- alcoholic extract or control using

deoxysugars (Keller Killiani Test) – 2ml of 10% gugo extract (This is

Place 10ml of the 80% ethanoic prepared by extracting 1g of gugo

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bark with 10ml of ethanol) in a solutions to the decanted

separate test tube. Add 10ml of supernatant. Precipitation at this

distilled water to each test tube, put point is indicative of a salting-out

stopper and shake the tubes reaction probably due to non-tannin

vigorously for 30 seconds. Allow to components. Filter off any

stand and observe over a period of precipitate. Add 3ml of the filtrate to

30 minutes. each of the three tubes.

F1. Gelatin Test

F. Screening for Tannins and Phenolic To test tube #1, add 2-3 drops

Compounds of a 1% gelatin solution; to test tub

Evaporate 100ml of 95% #2, add the same amount of gelatin

ethanoic extract to dryness on a salt reagent (1% gelatin, 10% sodium

steam bath. Remove the evaporating chloride); and to test tube #3, add

dish from the steam bath and add several drops of ferric chloride TS.

25ml of hot distilled water to the The absence of a reaction

residue. Mix well with a stirring rod with chloride indicates the absence

and allow it to cool at room of tannins and phenolic compounds.

temperature spontaneously. A greenish-blue or greenish black

Centrifuge the cooled extract for color after the addition of ferric

several minutes and decant the upper chloride is correlated with

half from each tube used. Add 3-4 precipitation on the gelatin-salt black

drops of 10% sodium chloride test indicating the presence of

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tannins of the catechol type. A blue and filter into small

black color after addition of ferric separatory funnel. Add 10ml

chloride is correlated with precipitate of benzene, shake to mix well

on the gelatin salt black test and allow the two phases to

indicating the presence of tannins of separate. Drain out the

the pyrogallol type. A negative aqueous layer (bottom layer)

gelatin-salt block test associated with and transfer the benzene

color production after the addition of phase (upper layer) to a test

the ferric chloride is indicative of the tube. Introduce 5ml of

absence of tannins and presence of ammonia, mix well and

other phenolic plant constituents. observe the benzene layer for

color change.

G. Screening for Anthraquinone Modified Borntrager test.

Heterosides Heat 0.3g of the plant powder with 10ml of

a. Borntrager test. Transfer 5ml 0.5 N potassium hydroxide and 1ml of dilute

of the ethanoic extract to an hydrogen peroxide for 10min. cool, filter

evaporating dish and dry over and acidify 5ml of the filtrate with

a steam bath. Defat the approximately 10drops of glacial acetic acid

residue in the dish with 5- and partition wit 10ml of benzene. Filter the

10ml of petroleum ether. Add benzene phase and transfer 5ml to a test tube

50ml of distilled water to the containing 2.6ml of ammonia TS. Mix well

defatted residue, mix well and observe for color changes.

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IN-VITRO MITOTIC 25
stopper on the tube, use cork

H. Screening for Cyanogenic Glycosides from which it is suspended in

a. Guignard Test. Place 2-5g of a piece of picrate paper. The

the crushed plant sample in a paper strip must not touch the

test tube. Moisten with water inner sides of the test tube.

and add a few drops of Warm the tube at 35-40⁰C or

chloroform to enhance keep it at room temperature

enzyme activity. Place a firm for 3 hours.

EXTRACTION PREPARATION Onions (Allium cepa L.) were

The crushed seed of Job’s tears (200 obtained from the local vegetable market at

gm) was soaked into 300ml ethanol for 3 San Carlos, Pangasinan, Philippines. 6

days in an incubator shaker (at 50 °C) to Onion is prepared into 6 Erlenmeyer flask.

obtain the ethanol extract. The solution was The first 4 flask is labelled as A1 A2 A3 A4

filtered and the filtrate was subjected to with concentration of 25%, 50%, 75% and

drying in oven at 70 °C so as to remove all 100% respectively. The 5th Erlenmeyer

the extraction solvent. The extract obtained Flask containing was distilled water is

was stored in refrigerator at 4 °C until labeled as B. the 6th Erlenmeyer Flask

further use. containing vitamin C labelled as C. the

onion bulb is placed on top of the

Preparation of the Alium cepa Erlenmeyer Flask . The onion bulbs were
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kept at room temperature for 14days over ANOVA was also utilized to determine if

until the roots have grown. there is significant difference in the length

of the onion seedlings depending on the

TOOLS FOR DATA ANALYSIS treatment applied or administered (p<.05).

ANOVA was also used in order to

The number of cell is counted using
determine whether there is significant
Electron Microscope and computed for its
difference in the number of dividing cells
Mitotic Index (MI). Using Clastogenicity
and the mitotic indices of the various media
bioassay, the anti-mitotic activity based on
used to cause or inhibit cell mitosis (p<.05).
microscopic analysis as to the mitotic index

should be equal to or lower than that of the Formula:

positive control vitamin c.

Mitotic Index = number of

dividing cells

To determine if the root

development is statistically different, one

1000
way Analysis of variance was conducted.
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IN-VITRO MITOTIC 27
Chapter 3 vitamin c which is 2.5 mm. The same result

RESULTS AND DISCUSSION was observe in treatment 2 (50% jobs tears

This chapter presents the data gathered, extract) and treatment 3 (75% jobs tears

analyzed and interpreted based on the extract) with 2mm and 2mm onion root

specific problems of the study. growing rate, which is lower than that of

Anti-mitotic Activity of jobs tears Extract vitamin C. Negative control has the least

through Macroscopic Analysis as to anti-mitotic activity for the onion seed

Inhibition of root growth germination has an average length of 5.5mm

In this analysis the different followed by 25% jobs tears extraction

concentration of jobs tears should inhibit the 2.5mm average root growing length

growth of the onion root at the same level of

or even greater than the positive control

vitamin c.

To have an anti-mitotic activity the average

length of the onion root must be equal to the

positive control. Table 1.a Macroscopic Analysis on the

As presented in table 1.a, where the Seventh Days of Onion root growing

root is treated with 4 different concentration Length

of jobs tears. With 100% jobs tears extracts

shows a greater anti-mitotic activity with an

average onion root growing length of 1

Flask with Replicates
.5mm as compared to the positive control
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Different (Onion root Average (negative

levels of growing (mm) control)

Concentration Length in

s mm)

Seco Anti-mitotic Activity of jobs tears Extract

First
nd through Macroscopic Analysis as to
Trial
Trial Inhibition of root growth

Flask 1 To consider the anti-mitotic activity

5mm 5mm 2.5mm
(25%) of the jobs tears extracts, the different

concentrations should inhibit the growth of

the onion roots. The length of growth in mm

that is permissible is equal to lower than the

Flask 2
2mm 2mm 2mm positive control (vitamin C) to consider its
(50%)
anti-mitotic effect.
Flask 3
2mm 2mm 2mm
(75%)
Table 1.b Macroscopic Analysis on the
Flask 4
2mm 1mm 1.5mm Fourteenth Days of Onion root growing
(100%)
Length
Flask 5

(positive 3mm 4mm 2.5mm

Flask with Replicates
control)
Different (Onion root Averag
Flask 6 6mm 5mm 5.5mm
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IN-VITRO MITOTIC 29
levels of growing e (mm) As gleaned from Table 1.b, 100% jobs tears

Concentration Length in extract has the greatest anti-mitotic activity

s mm) in terms of onion root growth inhibition,

Seco wherein, 100% jobs tears extracts shows a

First
nd greater anti-mitotic activity with an average
Trial
Trial onion root growing length of 1 .5mm as

Flask 1 compared to the positive control vitamin c

4mm 4mm 4mm
(25%) which is 2.5 mm followed by 75% jobs tears

Flask 2 extract (2mm) and 50% jobs tears extract

2mm 3mm 2.5mm
(50%) (2.5mm).

Flask 3 Anti-mitotic Activity of jobs tears Extract

2mm 2mm 2mm
(75%) through Microscopic Analysis as to

Flask 4 Clastogenecity Bioassay

2mm 1mm 1.5mm
(100%)

Flask 5 In order to say that the job tear’s

(positive 3mm 4mm 2.5mm extract has an anti-mitotic activity based on

control) microscopic analysis as to clastogenicity

Flask 6 bioassay, the mitotic index should be equal

(negative 6mm 6mm 6mm to or lower than that of the positive control

control) vitamin c. The lower the mitotic index the

lower the number of number of dividing

cells seen in the four phases of cell division

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IN-VITRO MITOTIC 30
(prophase, metaphase, anaphase and

teloaphase) the higher the antimitotic

activity.

As presented in Table 2, only 100% jobs

tears extract an anti-mitotic activity since the

mitotic index which is 0.015 is lower than

the Mitotic index of the positive control

vitamin C which is 0.033. On the other hand

other concentrations of jobs tears extract

such as 75%, 50% and 25% have mitotic

indexes of 0.33, 0.072, and 0.071,

respectively, which are lower than the

mitotic index of the positive control vitamin

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IN-VITRO MITOTIC 31
Concentration Stages of Mitosis Total Mitotic

Trial Index

Prophase Metaphase Anaphase Telophase

Number of

Cells

25%

1 10 10 8 5 33 0.033

2 12 12 7 7 38 0.038

Total 22 22 15 12 71 0.071

50%

1 13 11 6 5 35 0.035

2 14 11 8 4 37 0.037

Total 27 22 14 9 72 0.072

75%

1 7 6 3 2 18 0.018

2 6 4 3 2 15 0.015

Total 13 10 6 4 33 0.033

100%

1 3 2 2 1 8 0.008

2 2 2 2 1 7 0.007

Total 5 4 4 2 15 0.015
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Positive control

1 5 4 4 2 15 0.015

2 6 6 3 3 18 0.018

Total 11 10 7 5 33 0.033

Negative control

1 14 11 7 6 40 0.040

2 13 13 6 5 41 0.041

Total 27 24 13 11 81 0.081

positive negative
25% 50% 75% 100%
control control

1 5 2 2 2 3 6

2 5 2 2 1 4 5

n 2 2 2 2 2 2

X 5.000 2.000 2.000 1.500 3.500 5.500

s 0.000 0.000 0.000 0.707 0.707 0.707

Xave 3.250
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source Df SS MS F P-value

treatments 5 28.750 5.750 23.0000 0.0008

error 6 1.500 0.250

total 11 30.250

positive negative
25% 50% 75% 100%
control control

1 4 2 2 2 3 6

2 4 3 2 1 4 6

n 2 2 2 2 2 2

X 4.000 2.500 2.000 1.500 3.500 6.000

s 0.000 0.707 0.000 0.707 0.707 0.000

3.250

Xave
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source Df SS MS F P-value

treatments 5 26.750 5.350 21.4000 0.0009

error 6 1.500 0.250

total 11 28.250

Anti-mitotic Activity of jobs tears Extract through Microscopic Analysis as to

Clastogenecity Bioassay

positive negative
25% 50% 75% 100%
control control

1 0.033 0.035 0.018 0.008 0.015 0.040

2 0.038 0.037 0.015 0.007 0.018 0.041

n 2 2 2 2 2 2

X 0.036 0.036 0.017 0.007 0.017 0.041

s 0.004 0.001 0.002 0.001 0.002 0.001

Xave 0.025

source df SS MS F P-value
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treatments 5 0.002 0.000 90.2408 0.0000

error 6 0.000 0.000

total 11 0.002
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DISCUSSION Tannins

Based from the result of

It is one of the constituents that effect on
pytotochemical analysis, it was found that
morphology, cell proliferation and
the presence of Flavonoid’s, alkaloids and
apoptosis.
tannin. Sterols and triterpenses, steroid,

saponins, anthraquione heteroside and

glycosides were absent from the analysis. Job’s tears have anti mitotic activity in

the onion root tip (Alium cepa) lessen and

Alkaloids
inhibit the mitotic activity on the onion root
Alkaloids are the group of organic
(Alium cepa). From the result that we
compounds made up of carbon, hydrogen,
obtained, the total of different mitotic phase,
nitrogen and oxygen that are used to treat
trial 1 and trial 2 is computed. Prophase total
the cancer. The mechanism of this is that it
cell 6, Metaphase total cell 58, and
interacts with tubulin and disruption of
Anaphase total cell 39, and Telophase total
microtubule function, particularly of
cell 37. From these results we conclude that
microtubules comprising the mitotic spindle,
the cell division proceed usually in much
which causes metaphase arrest.
number is in the stage of prophase and

Flavonoids metaphase. The phase that is having less

number of mitotic phase is the anaphase and

Flavonoids are potent regulators of cyclin B
telophase stage. Between the anaphase and
and p21 for cell cycle progression, which
telophase phase, telophase is having lower
may play some roles in prevention of cancer.
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value. The effect of increasing concentration of 25% concentration which having 2.5mm

the Job’s tears extract. Experiment used different also, the 25% concentration is having same
concentration of Job’s tears extract, 25%, 50%, value with the positive control means that
75%, 100%. In 25% concentration the mitotic
has antimitotic activity. For the 50%, 75%
index of 0.033 trial 1, and 0.038 trial 2 total of
and 100% which is below the positive
0.071. In 35% concentration have a 0.035 trial 1
control value has also having antimitotic
and 0.037 trial 2 total of 0.072. In 50%
activity. The negative control has a greater
concentration 0.018 trial 1 and 0.015 trial 2 total
value than the positive control.
of 0.033. In 100% 0.008 trial 1 and 0.007 trial 2

total 0.015. From the result obtained upon

increasing the concentration of the Job’s tears For 14 days macroscopic

extract the more it lessen the mitotic activity in examination 25% concentration onion root

the root tip of the onion. length is 4mm, 50% concentration onion

root length is 2.5mm, 75% concentration

For the 7 days macroscopic examination
onion root length is 2mm, 100%
25% concentration onion root length is
concentration onion root length is 1.5mm.
2.5mm, 50% concentration onion root length
For the positive control Vitamin C onion
is 2mm, 75% concentration onion root
root length is 2.5 mm and for the negative
length is 2mm, 100% concentration onion
control which the distilled water onion root
root length is 1.5mm. For the positive
length is 6mm. Positive control which is
control Vitamin C onion root length is 2.5
2.5mm compare to the 25% concentration
mm and for the negative control which the
which having 4mm, the 25% concentration
distilled water onion root length is 5.5.
is having greater value than the positive
Positive control which is 2.5mm same to the
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control means that has less antimitotic antimitotic activity. The negative control has

activity. For the 50%, 75% and 100% which a greater value than the positive control.

is below the positive control value has more

CONCLUSION proven by studies. Hence, the preliminary

The results of the study proved that anti- can serve as groundwork for further studies

mitotic activity of Jobs tears, seed extract on in Genetics specifically in cellular division,

the root tip of the onion. Specifically the growth and related diseases.

anti-mitotic activity becomes more effective

as the concertation of the extract increases, RECOMMENDATIONS

however its anti-mitotic effect is more From the above conclusions the following

prominent in the later state of mitosis. are hereby recommended;

Furthermore, the effect of Jobs tears is 1. All doses and concentrations must be

statically evidence by a significant further subjected to more laboratory tests

difference as compared to result of the using more samples to more accurately

positive control using vitamin C. Most determine the claimed anti-mitotic property.

importantly, the mitotic activity is directly 2. A systematic research study of the Job

reduced by the presence of these active tear’s utilizing other essential parts.

components of Jobs tear’s i.e. Flavonoid’s, 3. The researcher recommends a parallel

alkaloids and tannin. In addition to these, study on the use of Job tear’s extract as an

these active components having anti mitotic alternative use to commercial drugs as anti-

property backed up by several literatures and mitotic.

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References

American Cancer Society. (2015). Cancer Facts & Figures Atlanta: American Cancer

Society

Arber, Agnes. (1965). The Gramineae--A Study of Cereal, Bamboo, and Grass. Wheldon

& Wesley, Ltd., New York.

Armstrong, W.P. (1994). Job's Tears. Ornament 18 (1): 104-105.

Armstrong, W. P. (1999). Job's Tears: A Wild Grass That Produces Nature’s Most

Perfect Bead. Retrieved from http://www2.palomar.edu/users/warmstrong/plapr99.htm

Arora, R.K. (1977). Job's Tears (Coix lacryma-jobi)--a Minor Food and Fodder Crop of

Northeastern India. Economic Botany 31 (3): 358-366.

Asis, Consuelo V., (1996). Plants of the Philippines (2nd Edition), Manila, Island

Publishing House, Inc.

Bolaños, A. B., (1997). Probability and Statistical Concepts: An Introduction, Rex

Book Store, Inc.

Cell Division and Cancer. (2014). In Nature Education. Retrieved from

https://www.nature.com/scitable/topicpage/cell-division-and-cancer-14046590

Duncan, R. (2006). Polymer Conjugates as anticancer Nanomedicines, Nature

Publishing Group, Vol. 8.

 VIRGEN MILAGROSA UNIVERSITY FOUNDATION
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IN-VITRO MITOTIC 40
Forni, G. (2006). Prevention of Cancer through Vaccination, Nature Publishing Group,

Vol. 7.

Guyton, A. and Hall, J. E. (2001). Textbook of Medical Physiology (10th Edition),

Philadelphia.

Heinrich, M., Barnes, J., Gibbons, S. and Williamson, E. M. (2012) Fundamentals of

pharmacognosy and phytotherapy. 2nd edition. Elsevier, Edinburgh, pp326. ISBN

9780702033889

International Agency for Research on Cancer (2012). Chemical Agents and Related

Components. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK304399

Job's tears (Coix lacryma-jobi). (2010). In Feedipedia. Retrieved from

https://www.feedipedia.org/node/472

Kerscher, A and Slack, F. J., (2006). mRNA Mutations, Nature Publishing Group, Vol. 8.

Laudico, A. V., Mirasol-Lumague, M., Medina, V., Mapua, C. A., Valenzuela, F. G., &

Pukkala, E. (2015). 2015 Philippine Cancer Facts and Estimates (pp. 1-79) (Philippines,

PHILIPPINE CANCER SOCIETY- MANILA CANCER REGISTRY).

Nature.com. (2014). Contents of Essentials of Cell Biology | Learn Science at Scitable.

Retrieved from http://www.nature.com/scitable/ebooks/essentials-of-cell-biology-

14749010/122997842 [Accessed 24 May 2017].

 VIRGEN MILAGROSA UNIVERSITY FOUNDATION
Martin P. Posadas Avenue, San Carlos City Pangasinan
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IN-VITRO MITOTIC 41
Normand, G., & King, R. W. (2010). Understanding Cytokinesis Failure. Advances in

Experimental Medicine and Biology Polyploidization and Cancer, 27-55. doi:10.1007/978-1-

4419-6199-0_3

OpenStax College (2016). Anatomy & Physiology. OpenStax CNX. Books

OpenStax College. (2013). Anatomy & physiology. Houston, TX: OpenStax CNX.

Retrieved from http://cnx.org/content/col11496/latest/

Pagoso, C. M., et.al. (2004). Introductory Statistics, Rex Book Store, Inc.

Phases of mitosis. (n.d.) In Khan Academy. Retrieved from

https://www.khanacademy.org/science/biology/cellular-molecular-biology/mitosis/a/phases-of-

mitosis

Schafer, K. A. (1998). The Cell Cycle: A Review. Veterinary Pathology, 35(6), 461-478.

doi:10.1177/030098589803500601

What Is Cancer?. (2015). In National Cancer Institute. Retrieved from

https://www.cancer.gov/about-cancer/understanding/what-is