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Vol. 5(6), 12-17, June (2016) Int. Res. J. Biological Sci.

Antimitotic Activity of Carica papaya Leaf Extract in the In Vitro

Development of Sea Urchin, Tripneustes gratilla Embryo
Pedro M. Gutierrez Jr.
Department of Biology, College of Arts and Sciences, Cebu Normal University, Cebu City, Philippines
[email protected]
Available online at: www.isca.in, www.isca.me
Received 19th May 2016, revised 1st June 2016, accepted 6th June 2016
Abstract
This study aims to determine the antimitotic activity of the ethanolic leaf extract of C. papaya in the sea urchin (Tripneustes
(Tripneust
gratilla)) embryo. The inhibition of cell proliferation of the crude extract in the sea urchin embryo was observed in three
different concentrations (0.5%, 1.00% and 1.5%) of the extract and two control groups. The time interval of each
developmental stage of sea urchin’s
rchin’s embryo treated with the different concentrations of the C. papaya extract was higher
compared to negative control group. Comparing the results in cleavage of the experimental groups, the lowest concentration
(0.50%) of the plant extract showed the fastest mitotic activity compared to other concentrations. On the other hand, the
highest concentration (1.50%) showed the slowest embryonic development compared to other treatment concentrations. In
addition, 0.50% concentration showed a comparable result with the positive control on the time interval during 2-cell2 and 4-
cell stages. C. papaya leaf extract showed antimitotic activity in the sea urchin embryos. The inhibition of sea urchin’s
proliferation in each developmental stage is dependent on the incr increase
ease plant extract concentration. The mitotic activity
inhibition of the various concentrations of the plant extract and the control group is also significantly different at 0.5 level
le of
significant. Tukey pairwise comparison test result showed that most ooff the compared treatment groups have a significant
difference between the time intervals of mitotic activity. In addition, the increasing concentration of the plant extract
increased the time interval between developmental stages. The antimitotic ability off the extract can be attributed to the
phytochemical component present in the plant. Previous studies revealed that phytochemical analysis of C. papaya contained
saponins, cardiac glycosides, and alkaloids. The phytochemical component of C. papaya is a goo goodd indicator that its extract is
suitable for a better pharmacological feature and a potent anti
anti-cancer agent.

Keywords:

Introduction addition,
ddition, some anticancer drugs work by inhibiting DNA
synthesis, thus preventing cell proliferation10.
The World Health Organization stated that approximately 80%
of the human population in rural region are benefited by the The medicinal effect of plant relies mainly on its natural
herbal medicines for healthcare needs1. Many medicines products. Plants produce phytochemicals to its entire parts as a
including strychnine, aspirin, vincristine, and taxol are of herbal means of protection and aid for survival11. Since plants are
plant origin2. Philippine studies firmly established that plants stationary, they have to cope with the challenges in the
such as as Meliadubia3. Moringaoleifera era4, Dichapetalum environment to compensate their lack of movement through the
5 6
gelaniodes , and Cucurbita moschata , are among of the aid of substances called secondary metabolites12. Secondary
presently considered cancer cell division inhibitors with tumor metabolites are not directly involved in the growth and
cytotoxic activity. reproduction of a plant; instead, they provide adaptations that
are necessary for the plants’ survival13). Secondary metabolites
Cancer is one of the significant health problems affecting tend to be unique to specific families and genera14. These
citizens worldwide. Each year, it is estimated that 6 million compounds give color to the plant helps the plant respond
respon to its
people are affected with cancer diseases. In the Philippines, environment and protects itself from radiation15. It also acts as a
approximately 200,000 Filipinos are affected by cancer pain defense by way of secreting substances that are toxic to
despite of some available methods for its treatment7. The herbivores and microorganism that attack the plant16. It is the
scientific community continuously explores new compounds protective role of secondary metabolites in plants that are ar
that can be used as anticancer agents that are more effective and considered as an underlying reason for plants having its
safe8. One of most successful chemotherapeutic compounds therapeutic effect. Hence, finding a plant that has the capability
currently used for anti-cancer
cancer treatment that interfere with the of treating cancer based on its secondary metabolites
normal progression of mitosis are derived from plants 9. In composition is an advantage for finding the subject plant.

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The search for an appropriate plant for the study is considered The female sea urchins were placed in the 100 mL beaker filled
not only the phytochemical component, but also its traditional with 100 mL filtered seawater to shed its eggs. The eggs were
use. Carica papaya, commonly known as papaya is a tropical examined under the microscope to check for any abnormalities
plant with a fruit viewed as orangre-red, yellow-green and and whether the eggs are mature or not. Mature eggs were
yellow-orange hues. The whole plant parts are known to determined by the presence of small nucleus in the periphery of
possess medicinal properties for the cure of warts, sinuses, the cell membrane and a large amount of cytoplasm23.
eczema and also injected into indolent glandular tumors17.
Young leaves of C. papaya is reach of the following In the fertilization process, the 100 mL egg solutions were
phytochemicals: flavonoids, alkaloids, phenolic compounds and pooled in the 1 L beaker with 2 mL sperm suspension. The eggs
cynogenetic compounds18. were swirled and allowed to settle for 15 minutes. About 500
mL of water and other mixtures were drained off; the washed
Sea urchin is a widely studied organism which records profound eggs were transferred to 100 ml beaker. The 100 ml eggs were
biological information, readily available, and reproducible at transferred again to 500 mL filtered seawater. The twice
low laboratory cost19. Sea urchin provides a suited system for repetition of egg suspension removed the egg’s jelly coat24.
test organism because of its capability to induce artificial
spawning, fertilization and embryo transparency20. Sea urchin The embryos were exposed to an increasing concentration from
and human are closely related to their genes because they share 0.5%, 1.0%, and 1.5% of C. papaya extract. The test was
a similar ancestor, deuterostome, the phylum of animals that measured by determining how long the embryo transformed
includes echinoderms and chordates21. Most importantly, the from one cell to two-celled cleaving embryo and quantifying the
fertilized sea urchin bioassay determines the potential inhibitory number of cells developed in every cell stage at a certain time
in DNA synthesis to be used as an anticancer remedy22. If the interval.
cells exhibit cytotoxicity on the tested plant this implies cells
antimitotic activity. This study aims to determine the antimitotic Sea Urchin Bioassay: The mitotic assay was adapted from
activity of C. papaya leaf extract on the development of the sea Militão et al. with some modifications. One (1) mL of sperm
urchin (Tripneustes gratilla) embryo. suspension was added to 100 ml filtered seawater containing the
eggs. The union of sperm and egg were viewed under the
Materials and Methods microscope. The appearance of fertilization membrane marks
the onset of fertilization. When the eggs were already fertilized,
Preparation and Extraction of Plant Samples: The collected 5 mL of egg and sperm suspension were distributed into the 15
plant samples were washed with tap water and rinsed with petri dishes designated with their assigned treatments. The
distilled water. It was then air-dried at room temperature. The experimental group received three various concentrations C.
dried samples were cut and pulverized into small pieces and papaya extract (0.50%, 1.0%, and 1.5%) into the 9 petri dishes.
ground using electric blender. The powdered plant samples were The positive control group used colchicine drug applied to the 3
soaked with 100% ethanol for 48 hours. After which, the petri dishes and another 3 petri dishes for the negative control
samples were filtered and the filtrate was concentrated in a group. Each microscope was carefully viewed to determine the
rotary evaporator. minute interval of cell division from the first cell-stage until 32-
cell stage. After an hour, a sample of 1 ml solution was
Sexing and Spawning of Sea Urchin: Adult sea urchins T. collected from every concentration combined with 1 mL of 5%
gratilla with a diameter of 10-15 cm were purchased from a formaldehyde in a test tube to preserve for later analysis. A total
local fisherman from Marigondon, Lapu-lapu City, Philippines. of 100 cells were scored for counting the number of cells.
Sea urchins were placed in a box plastic container filled with
seawater and sea grasses for their food with an aerator for Data Analysis: The following statistical tools were used to
oxygen supply. Identifying the sex of T. gratilla was done by analyze the data: Arithmetic mean to get the average percentage
injecting of 0.5 ml of 0.5M KCl through the periostomal of inhibition. One-Way Analysis of Variance (ANOVA) was
membrane was used to determine the sex of sea urchin. The used to determine the significant difference between the control
female T. gratilla secretes orange eggs while males release and the different concentrations of C. papaya. The 0.05 level of
cream-colored semen. Viewing under the microscope also probability was used as a comparison for significance. Tukey’s
validated the sex of sea urchin. pairwise comparison tests to determine which groups differ
significantly from the other group by its time interval.
Spawned male sea urchins were transferred to 50 mL beaker
with 1 mL filtered seawater and were placed in the water bath Results and Discussion
filled with ice for the continued shedding and preservation of
sperm. A small amount of sperm was placed into the slide with Table-1 shows the mean time interval measured in minutes of
2 drops of filtered seawater and viewed under a microscope to cleavage in sea urchin treated with the three various
test for its motility. The sperm with high motility was used for concentrations of C. papaya extract and the control groups. In
sperm pooling to be placed into 100 mL beaker. the negative control, normal cell division was observed with the

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International Research Journal of Biological Sciences ________________________________________________ISSN 2278-3202
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shorter time difference in every developmental stage which delay of the development of each stage could be due to the
reaches up to 32-cell stage as compared to the various disturbance of the cytoskeletal structures, which are critical for
concentrations of the plant extract and the positive control embryonic development. These structures establish the internal
(Colchicine). In the experimental group, 0.5% concentration has structure of the cell, which is essential for maintaining its
the fastest rate of cell division but shows very different result of normal functions25.
the time interval between 2-cell and 4-cell stage in the negative
control. Meanwhile, the highest concentration of plant extract Table-2 and 3 present the one-way analysis of variance
(1.50%) revealed the slowest rate of cell division that divides (ANOVA) for time interval at 2-cell stage and 4- cell stage of
only until 2-cell stage. The negative control revealed the fastest sea urchin embryo development treated with the various
rate of cell division as compared to the various treatment concentrations of the C. papaya extract and the control groups.
concentrations of the plant extract while positive control treated Result reveals that there is high significant difference of the
with colchicine was comparable to the result gathered in the time interval in 2-cell and 4-cell developmental stages of sea
0.5% concentration. urchin embryos treated with the three various concentrations of
the plant extract along with the control group. The delay and
Comparing the three different concentrations treated with C. inhibition of the cell division could be due to the disturbance of
papaya ethanolic extract, 0.5% concentration revealed the the cytoskeletal structures, which are critical for embryonic
fastest rate of cell division while 1.5% concentration revealed development and essential for maintaining its normal
the slowest rate of mitotic activity. Results also revealed that the functions25. Semenova et al. also stated that the disruption of the
sea urchin embryos treated with the various concentrations of cell division could be due to the disturbance of the tubulin.
the plant extract manifest mitotic activity from 2-cell to 4-cell Tubulin is a major protein in microtubule, cell organelle whih
stage only contrary to the negative control which proliferate up play significant role in mitosis specifically in the formation
to 32-cell stage. This implies that the mitotic activity inhibition of mitotic spindle and separation of chromosomes during
of the plant extract is concentration-dependent. anaphase. Thus, deactivating tubulin in rapidly proliferating
tumor cells is an effective way for cancer therapy25.
Result shows inhibition of mitotic activity in T. gratilla embryos
treated with different concentrations of C. papaya extract. The

Table-1
Mean time interval of the early embryonic developmental stages of sea urchin eggs treated with the various concentrations
of the C. papaya extract
Mean Time Interval of Cleavage (minutes)
Treatments
2-cell stage 4-cell stage 8-cell stage 16-cell stage 32-cell stage

Negative Control 44.75 31.00 54.75 38.75 27.25

Positive Control
48.50 75.25 -- -- --
(Colchicine)
0.50% 53.50 92.00 -- -- --

1.00% 61.25 178.00 -- -- --

1.50% 69.75 -- -- -- --

Table-2
One-Way Analysis of Variance (ANOVA) for time interval at 2-cell stage of sea urchin embryo development exposed to
C. papaya extract and the control groups
Source SS df MS F Fcritical p-value

Between 2421.7 4 605.43 29.557 3.0556 0.0000

Within 307.25 15 20.483

Total 2728.95 19

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Table-3
One-Way Analysis of Variance (ANOVA) for time interval at 4-cell stage of sea urchin embryo development treated with
the various concentrations of the C. papaya extract and the control groups
Source SS df MS F Fcritical p-value

Between 43757.2 2 21879 17.849 4.2565 0.0007

Within 11031.8 9 1225.8

Total 54788.9 11

Table-4
Tukey Pairwise comparison test of time interval in various developmental stages between the different treatments
(Positive control, negative control, 0.5%, 1.0%, 1.50%)
P-value of Developmental Stages
Grouped compared
2-cell 4-cell 8-cell 16-cell 32-cell

Positive vs. Negative *0.0175 *0.0120 ----- ----- -----

Positive vs. 0.50% 0.1009 0.5959 ----- ----- -----

Positive vs. 1.0% *0.0085 *0.0001 ----- ----- -----

Positive vs. 1.50% *0.0000 ----- ----- ----- -----

Negative vs. 0.50% *0.0257 *0.0244 ----- ----- -----

Negative vs. 1.0% *0.0427 *0.0000 ----- ----- -----

Negative vs. 1.50% *0.0000 ----- ----- ----- -----

0.50% vs. 1.0% 0.1197 *0.0300 ----- ----- -----

0.50% vs. 1.50% *0.0011 ----- ----- ----- -----

1.0% vs. 1.50% *0.0366 ----- ----- ----- -----

*implies that the significance lies in the compared group.

To pinpoint statistically which groups differed significantly on The following are the functions of the phytochemicals present in
its time interval at the specific stage, Tukey Test was further the plants that mainly interacts with the disruption of
employed considering the consistency of the replicates. Results microtubule and cell cycle. Saponin has relationship with sex
(Table-3) revealed that the compared treatment groups differ hormones like oxytocin. Oxytocin is a sex hormone involved in
significantly in the 2-cell stage except for positive control controlling the onset of labour in women and the subsequent
versus 0.50% and 0.50% versus 1.00% concentrations. In release of milk27. Alkaloids bind to the building blocks of a
addition, at 4-cell stage, the compared treatment groups differ protein called tubulin, during cell division and inhibiting its
significantly except for positive control versus 0.50% formation28. Steroids block the G2/M phase of cell cycle, induce
concentration. apoptosis, and change of Ca2+ distribution that triggers the
cytoplasmic event breakdown in somatic cells29. Terpenoids
The Phytochemical analysis of C. papaya showed that the blocks mitosis by stabilizing tubulin polymers and thereby
leaves contained saponins, cardiac glycosides, and alkaloids26. inhibiting disassembly of microtubules30. Glycoside disrupts the
Tannin was absent in the leaves. The presence of saponins appearance of mitotic-spindle which leads to abnormalities
supports the fact that papaya leaf has cytotoxic effects such as during anaphase stage31.
permealization of the intestine as saponins are cytotoxic27.

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Sea urchin embryogenesis was known to have well-defined .int/health_technology/book_who_traditional_medicine_str

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