In Vitro Mitotic Inhibition of Job's Tears

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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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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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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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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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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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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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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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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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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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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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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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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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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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

COLLEGE OF MEDICINE
IN-VITRO MITOTIC 23
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

COLLEGE OF MEDICINE
IN-VITRO MITOTIC 24
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.

COLLEGE OF MEDICINE
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
COLLEGE OF MEDICINE
IN-VITRO MITOTIC 26
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.
COLLEGE OF MEDICINE
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
COLLEGE OF MEDICINE
IN-VITRO MITOTIC 28
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
COLLEGE OF MEDICINE
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

COLLEGE OF MEDICINE
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

COLLEGE OF MEDICINE
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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
COLLEGE OF MEDICINE
IN-VITRO MITOTIC 32
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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IN-VITRO MITOTIC 34
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
COLLEGE OF MEDICINE
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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.
COLLEGE OF MEDICINE
IN-VITRO MITOTIC 37
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
COLLEGE OF MEDICINE
IN-VITRO MITOTIC 38
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.

COLLEGE OF MEDICINE
IN-VITRO MITOTIC 39
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VIRGEN MILAGROSA UNIVERSITY FOUNDATION

Martin P. Posadas Avenue, San Carlos City Pangasinan

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

Penaverde, Kayle Paolo S. Pinlac, Prabin Silwal

for the interpretation of the results.

process of treating of cancer.

Tears (Coix lacryma-jobi)

from cancer annually. 13 out of 100 males

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

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

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

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

division is the feeding mechanism of cancer

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

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

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

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

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

primarily segmented into three sequential

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

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

visible chromosomes. Because during chromatids, with their connected

cell receive its own complement of DNA,

capable of growing and dividing again while

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

surprising that cytokinesis can sometimes

fail. Cytokinesis failure leads to both

suppressor genes or oncogenes that directly

responsible for its Anti-mitotic activity.

treatment for cancer. Namely, they are

surgery, chemotherapy, radiation therapy,

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

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

biochemical components with potential rapidly multiplying cancer cells. Radiation

shrink tumors or eliminate cancer cells. It

of Health stated 20-30 different theories on

our body to prevent heart diseases. In fact,

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

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

2000). researches the studies was utilized the seed

parts to obtaining the extract solvent that

readings. is placed in distilled water which serves as a

negative control and labelled as B. The 6th

crushed. The crushed seed sample is brought

STATEMENT OF THE PROBLEM

This research aims to determine and

tear using onion root as to the

3. Is there significant different on the

following concentration to the

positive and negative control

There is no significant inhibition Job’s

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

the Alium cepa roots.

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

anti-mitotic activity. towards cancer research and drug

development for the treatment of cancer.

deemed to be significant in providing Professionals: The vital information

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