Philippine Science High School

Eastern Visayas Campus

Mitodepressive Effect of Methanolic Extract of

Water Hyacinth (Eichhornia crassipes) Leaves
Using Mung Bean (Vigna radiata) Bioassay

Rex Angelo M. Jusayan

David Anthony C. Canillas
March 2014

Mitodepressive Effect of Methanolic Extract of

Water Hyacinth (Eichhornia crassipes) Leaves
Using Mung Bean (Vigna radiata) Bioassay

by
Rex Angelo M. Jusayan
David Anthony C. Canillas

Submitted to the Faculty of the

Philippine Science High School Eastern Visayas Campus
in partial fulfillment of the requirements for
Science and Technology Research 2
March 2014

ABSTRACT
The methanolic extract of water hyacinth (Eichhornia crassipes) leaves was evaluated
for its mitotic inhibition activity through mung bean bioassay. The study was done to
discover an alternative herbal medicine that would be effective in treating cancer cell
metastasis.
Powdered water hyacinth leaves were extracted using soxhlet extractor and the
solution was concentrated through simple distillation. Eighteen mung beans were exposed to
each of the experimental setups (10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL), to the
distilled water and to the copper (I) chloride setups. After 24 and 48 hours of exposure to the
treatments, the seeds were weighed and the percentage of imbibition inhibition was
calculated, as suggested by G. Satyanarayana Murthy, et al. The seedling length and radicle
length retardation were also examined after 1 week of sprouting.
Water hyacinth leaf extract (10 mg/mL, 20 mg/mL, 30 mg/mL and 40 mg/mL) did
not induce dose dependent reduction in both radicle and seedling length. All the
concentrations of water hyacinth leaf extract has been found out to exhibit significant radicle
and seedling lenght reduction (p<0.05 & p<0.001). The percentage imbibition of the seeds
was also inhibited by water hyacinth leaf extract (10 mg/mL, 20 mg/mL, 30 mg/mL, 40
mg/mL) by 49.34%, 65.08%, 74.20% and 27.30%, respectively.
These morphometric analyses therefore indicate that water hyacinth leaf extract can
cause mitodepression in mung bean and thus, it can be used as potential treatment for cancer
cell progression.

APPROVAL SHEET
This research work entitled, Mitodepressive Effect of Methanolic Extract of
Water Hyacinth (Eichhornia crassipes) Leaves Using Mung Bean (Vigna radiata)
Bioassay by Rex Angelo M. Jusayan and David Anthony C. Canillas, presented to the
Faculty of the Philippine Science High School Eastern Visayas Campus in partial
fulfillment of the requirements in Science and Technology Research 2, is hereby accepted.

Dr. Estrella E. de Dios

Research Adviser

Ana Maria A. Chupungco

Research Adviser

ACKNOWLEDGEMENTS
This research has undergone many adjustments, challenges, problems, and failure.
Everything this research has gone through was undeniably rough. We had to make many
transformations in our procedure. Even the title was changed several times. We have tried
different procedures before settling down to the simplest ones so we could finish it on time.
But despite having all those undertakings, we still would like to express our gratitude to God,
to our parents, and to our research advisers.
First and foremost, we would like to express our gratitude to God. He is the source of
our strength and knowledge. It is also because of Him that we are still alive. Despite the
catastrophic storm, Yolanda, which hit our hometown, he is still there to guide us and help us
to stand up once again.
Secondly, we would like to thank our ever-supportive parents for giving us all the
financial aid we needed. Though the budget that they give was admittedly limited, we are
still thankful because through that, we were able to learn how to maximize every resource
that we had.
We would also like to thank our research advisers, Dr. Estrella de Dios and Ms. Ana
Chupungco, for guiding us to every procedure we were doing, to finishing the research paper,
and to looking for possibilities of our study.
Lastly, we would like to convey our sincerest gratefulness to our research buddy,
Genevieve Llanita, who was taken by the chaotic remarks of Typhoon Yolanda. Even though
she was not able to be with us until the end of the project, she had contributed so much in the
study. We would have not been able to conquer this task if it had not been because of her.
To the reader, we hope that youll find this study knowledgeable. =)
ii

TABLE OF CONTENTS
Page

Approval Sheet........................................................................................................................... i
Acknowledgements ................................................................................................................... ii
Table of Contents ..................................................................................................................... iii
List of Tables ............................................................................................................................ v
List of Figures .......................................................................................................................... vi
Introduction ............................................................................................................................... 1
Background of the Study .............................................................................................. 1
Statement of the Problem .............................................................................................. 2
Significance of the Study .............................................................................................. 2
Scope and Limitations................................................................................................... 3
Review of Related Literature .................................................................................................... 5
Cancer ........................................................................................................................... 5
Antimitotic .................................................................................................................... 6
Mung Bean Assay ......................................................................................................... 6
Water Hyacinth ............................................................................................................. 7
Biological Properties of Water Hyacinth ...................................................................... 8
Materials and Methods ............................................................................................................ 10
Process Flowchart ....................................................................................................... 10
Extraction of Water Hyacinth ..................................................................................... 11
Preparation of Treatments ........................................................................................... 11
Mung Bean Assay ....................................................................................................... 11
Results and Discussion ........................................................................................................... 13
iii

Recommendations ................................................................................................................... 18
Bibliography ........................................................................................................................... 19
Appendices .............................................................................................................................. 21
Raw Data .................................................................................................................... 21
Statistical analysis ..................................................................................................... 23
Documentation ........................................................................................................... 26

iv

List of Tables
Table

Title

Page

Effect of water hyacinth leaf extract on seedling and shoot growth of Vigna
1

13
radiata
Effect of the treatments on the mean weight and percent imbibition

14
inhibition of Vigna radiata at T-0 and T-1
Percentage imbibition inhibition of some synthetic and plant extract

15
anticancer drugs

List of Figures
Figure

Title

Page

Cancer growth Progression

05

Response of Cell to Antimitotic Drug Treatment

06

Water Hyacinth

07

Effect of water hyacinth leaf extract on seedling and radicle growth (mm) of
4

13
Vigna radiata at 168h treatment

vi

INTRODUCTION
Background of the Study
In the past few years, global burden of cancer continues to increase dramatically. In
2007, more than 12-million people were diagnosed with cancer and in 2008, there was an
estimated of 12.7 million cancer cases and 7.6 cancer deaths around the world. This figure is
expected to escalate 21 million by 2030 (Gascoigne and Taylor, 2009) (World Cancer
Research Fund International, n.d.). According to the American Cancer Society, at least one
third of these individuals are not expected to survive the disease, making cancer the second
most prevalent cause of death worldwide. Systemic chemotherapy forms the backbone of
cancer treatment, and agents that suppress mitotic spindle assembly, so called anti-mitotics,
are usually used to treat different types of cancer.
Eichhornia crassipes, or commonly known as water hyacinth, is considered as one of
the most invasive aquatic plants in the world. The plants bulky leaves shield the flora and
fauna under it from the suns rays which is essential for their life. The weed forms a dense
mat that interferes with the flow of water in irrigation canals causing flood. This dense mat
also serves as a breeding ground for mosquitos. Water hyacinth also competes with rice soil
nutrients reducing the yield when left uncontrolled.
Despite the plants destructiveness, it also contains useful biological components
which can be beneficial to humans. The plant possesses abundant phytochemicals and
antioxidants (K. Vasu, J.K. Goud, A. Suryam and M.A.S. Charya, 2009) (A.R. Kurup et al.,
2013) which are important properties that can inhibit the mitotis of cancer cells (A.
Thenmozhi, A. Nagalakshmi and U.S.M. Rao, 2011) (R.W. Kehr, 2013) (C.L. Hernandez, I.
Villasenor, E. Joseph and N. Tolliday, 2008) (Patil S., Narayanan S., Eibl G., and Jolly C.L.,
1

2004) (S. Bhattacharya & P.K. Haldar, 2012) (L. Dobjanschi et al., 2008). An antimitotic
substance can be used as a treatment for cancer because it can disrupt microtubules, which
are the structures that pull the cell apart when it divides. Through the mechanism of the said
drug, metastasis can be suppressed.
Statement of the Problem
Main Problem
Does water hyacinth (Eichhornia crassipes) leaf extract have mitodepressive effect
using Mung Bean (Vigna radiata) Bioassay?
Sub-Problems
1) Will there be a significant, among the following concentrations, capability on
increasing the percentage of imbibition inhibition after 24 and 48 hours of
imbibition?
a) 10 mg extract / 1 mL distilled water
b) 20 mg extract / 1 mL distilled water
c) 30 mg extract / 1 mL distilled water
d) 40 mg extract / 1 mL distilled water
2) Will the water hyacinth extract (10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL)
cause seedling and radicle retardation on mung bean after 168 hours (1 week) of
sprouting?
Significance of the Study
Today, when there are already reports on the drastic increase in the occurrence of
cancer diseases, searching for new sources of biological components which can inhibit the
spread of cancer cells, is an issue.
2

Antioxidants and phytochemicals such as alkaloids, ellagic acids, phenols, steroids,

tannins, triterpenoids and saponins are potential components in plants which can help treat
cancer (A. Thenmozhi, A. Nagalakshmi and U.S.M. Rao, 2011) (R.W. Kehr, 2013) (C.L.
Hernandez, I. Villasenor, E. Joseph and N. Tolliday, 2008) (Patil S., Narayanan S., Eibl G.,
and Jolly C.L., 2004) (S. Bhattacharya & P.K. Haldar, 2012) (L. Dobjanschi et al., 2008).
Water hyacinth, an aquatic invasive plant, has been found out to contain these components
(K. Vasu, J.K. Goud, A. Suryam and M.A.S. Charya, 2009) (A.R. Kurup et al., 2013) and so,
the present study aims to evaluate the antimitotic effect of the said plant.
Water hyacinth is common and easily attainable. And so, if proven effective as
antimitotic agent, people can have an additional alternative and cheaper way of dealing with
cancer.
Also, the said plant overpopulates the ecosystem and threatens the lives of other
organisms especially the plant species. And so, this study would indirectly help in
minimizing the population of the said invasive plant since the people would already be
motivated to make use of this plant. In this way, the negative attribute of water hyacinth
would be outweighed by its positive ones.
Scope and Limitations
This study is only limited to the evaluation of the antimitotic property of water
hyacinth (Eichhornia crassipes ) leaf extract. Human testing will not be conducted since the
researchers will only use bench-top bioassay, Mung Bean (Vigna radiate) Bioassay, in
assessing the plants antimitotic property.

The study is not going to isolate a specific phytochemical from the extract. Pure crude
extract, containing the synergy of all the phytochemicals extracted (alkaloids, ellagic acid,
phenols, steroids, tannins, triterpenoids and saponins), would be used.

REVIEW OF RELATED LITERATURE

Cancer
Cancer is a group of ailments characterized by out-of-control progression of aberrant
cells. There are over 100
different forms of cancer, and
each is classified by the type
of cell that is initially affected
(MediLexicon

International

Ltd, n.d.)
Figure 1: Cancer growth Progression
From: www.physio-pedia.com

Cancer cells originate

from normal cells when their deoxyribonucleic acid (DNA) inside the cell nucleus is
impaired. DNA is present in every cell and it directs the entire cells actions, growth, death,
protein synthesis and many more. When DNA is impaired in a normal cell, the cell either
repairs the damage or the cell dies. In cancer cells, the damaged DNA is not repaired, and the
cell does not die. Instead it gives rise to more such aberrant cells with aberrant DNA. These
new cells all have similar faulty DNA of the original cancer cell (Mandal, 2013). Tumors
may from as the cells proliferate. Not all of these tumors are cancerous, however. Those that
are cancerous are called malignant tumors and those that are not, are called benign tumors.
DNA damage may be inbred from parents or may be a spontaneous problem that
occurs during the lifetime of a person. DNA impairment may also be generated by exposure
to certain environmental toxins. There are, however, numerous factors that may cause cancer
and it is tough to pin point an exact cause (Mandal, 2013).
5

Antimitotic
When a tumor successfully spreads to other parts of the body and grows, invading
and destroying other healthy tissues, it is said to have metastasized. This process itself is
called metastasis, and the result is
a serious condition that is very
difficult to treat.
Drugs that disrupt mitotic
progression

are

commonly

referred to as anti-mitotics or
mitotic-inhibitors. These drugs
interrupt microtubules, which are
structures that pull the cell apart

Figure 2: Response of Cell to Antimitotic Drug Treatment

From: http://jcs.biologists.org/content/122/15/2579/F1.expansion.html

when it divides. Mitotic inhibitors are used in cancer treatment, because cancer cells are able
to

grow

and

eventually

spread

through

the

body

(metastasize)

through

continuous mitotic division and cancer cells are more sensitive to inhibition of mitosis than
normal cells (Gascoigne and Taylor, 2009).
Mung Bean Assay
There are a variety of methods for screening the antimitotic activity of different
substances. These include the animal model system for inhibition of tumor growth implanted
in mice, potato disc tumor induction assay and the inhibition of proliferation of cancer cell
lines in in vitro cultures. However, these methods are expensive and require expensive
infrastructure.

In the study conducted by G. Satyanarayana Murthy, T.P. Francis, C. Rajendra Singh,

H.G. Nagendra and Chandrashekhar Naik (2011), entitled An assay for screening antimitotic activity of herbal extracts, an inexpensive but reliable assay using the measurement
of mitotic index of sprouting seeds has been discovered.
The method is useful in screening the claims and counterclaims in the use of
medicines, herbal extracts, etc. as antimitotic agent. In fact, asparagus, which is a wellknown alternate food supplement for cancer cure has inhibited the sprouting of mung beans
(G. Satyanarayana Murthy, T.P. Francis, C. Rajendra Singh, H.G. Nagendra and
Chandrashekhar Naik, 2011). However, the molecular mechanisms by which sprouting is
affected by antimitotic drugs are not known. It may be due to the interference in the cell
cycle or through interference in spindle formation. Comparative biochemistry of sprouting of
seeds and neoplastic growth in humans is yet to be studied to understand the biochemical
basis of specificity of the assay (G. Satyanarayana Murthy, T.P. Francis, C. Rajendra Singh,
H.G. Nagendra and Chandrashekhar Naik, 2011).
Water Hyacinth
Water hyacinth is a free-oating
perennial aquatic plant that is considered
the worlds worst aquatic weed (Wersal
and Madsen, 2012). It can reproduce by
both sexual and asexual means, though
asexual (vegetative) propagation has been
largely

attributed

to

its

widespread

Figure 3: Water Hyacinth

From: http://sites.duke.edu/writing20_12_f2010/2010/09/13
/water-hyacinth-brought-down-by-climate-pattern-or-tinybeetle/

distribution. Water hyacinth effectively doubles the number of plants within 12.5 days
(Penfound and Earle, 1948).
The water hyacinths stem is upright and bears the flowers. The flowers are blueviolet or lilac, and attractive. They have six petals and one endures a yellow spot. The leaves
are green to dark-green, round to oval, and glossy with leathery blades. The leafstalks
(petioles) are thick and spongy and contain some amounts of air that enable the plant to float.
Masses of fine branching roots hang underneath the floating plant.
With its bulky leaves, it shields the flora and fauna under it from the suns rays which
is essential for their life. The plant is propagated by seeds and vegetative off radicle. The
weed forms a dense mat that interferes with the flow of water in irrigation canals causing
flood. This dense mat also serves as a breeding ground for mosquitos. Water hyacinth also
competes with rice soil nutrients reducing the yield when left uncontrolled (PAN Germany,
n.d.).
Even if this aquatic plant is destructive, water hyacinth also possesses some
biological properties that are beneficial to the people, such as being antibacterial and
antifungal in the study of Baral and Vaidya (n.d.) entitled Biological and Chemical
Assessment of Water Hyacinth (Eichhorna Crassipes (Marts.) Solms.) Ofphewa, Lake Nepal.
Biological Properties of Water Hyacinth
Being the worlds worst aquatic weed, great attention has been focused on controlling
water hyacinth. As part of this, research has sought to use the said plant for benecial
purposes (Wersal and Madsen, 2012) (Rao and Nagavani, 2010).
In a study of K. Vasu, J.K. Goud, A. Suryam and M.A.S. Charya (2009) entitled,
Biomolecular and Phytochemical Analyses of Three Aquatic Angiosperms, the methanolic
8

extract of Eichhornia crassipes was screened for the presence of various phytochemicals by
standard procedures. The present study indicates that the plant contains alkaloids, ellagic
acid, phenols, steroids, tannins, triterpinoids and saponins.
In another study conducted by A.R. Kurup et al. (2013), entitled Detailed Analysis
on Phytochemicals, Antioxidants, Antimicrobial Activity of Eichhornia Crassipes, the
antioxidant activity of water hyacinth leaf was screened. Using two different methods,
reducing power assay and DPPH assay, the study confirmed the effectiveness of the said
plant as antioxidant agent.
As stated by different researchers, the pronounced antimitotic and anticancer
activities of plant extracts was due to their potential antioxidant property especially by the
key role of phytochemicals such as alkaloids, ellagic acid, phenols, steroids, tannins,
triterpenoids and saponins (A. Thenmozhi, A. Nagalakshmi and U.S.M. Rao, 2011) (R.W.
Kehr, 2013) (C.L. Hernandez, I. Villasenor, E. Joseph and N. Tolliday, 2008) (Patil S.,
Narayanan S., Eibl G., and Jolly C.L., 2004) (S. Bhattacharya & P.K. Haldar, 2012) (L.
Dobjanschiet al., 2008). And so, since the extract of water hyacinth contains these important
phytochemicals (K. Vasu, J.K. Goud, A. Suryam and M.A.S. Charya, 2009) and powerful
antioxidants (A.R. Kurup et al., 2013) there is a huge possibility that the extract of water
hyacinth also has an antimitotic activity.

MATERIALS AND METHODS

Process Flowchart

Treatment
Preparation

Subject
Preparation

Weigh mung beans

(85.0 4.0 mg)

Air dry the water

hyacinth leaves

Put 6 seeds into

each well of a 6well microtiter
plate

Turn to powder
using mortar &
pestle

Concentrate
through simple
distillation

Extract using
soxhlet apparatus

Prepare different
concentrations of
the extract

Weigh each bean

and calculate for
% imbibition
inhibition after 24
and 48 hours

Prepare the
positive (cuprous
chloride) and
negative (distilled
water) controls

Measure radicle
length and
seedling length
after 168 hours (1
week)

10

Extraction of Water Hyacinth

Water hyacinth leaves were collected in Calbayog City, Samar. The root and shoot
portions of the plant were cut off and the leaves were washed thoroughly in tap water to free
from debris. The leaves were blot dried using paper towel to remove excess water. Only
leaves that were free from dark spots were used in the experiment. The leaves were air-dried
for 2 weeks under shade and then turned into powder using mortar and pestle.
Dry powdered leaves were used for extraction. Three soxhlet extractors, each
containing 16 grams of the plant material and 150 mL of methanol, were used for extraction.
The resulting solution was subjected to simple distillation to remove the solvent. The
concentrated solution was then poured into a beaker, covered with aluminum foil and then
stored at 4oC for further investigations (Vasu, K. Et al, 2009).
Preparation of Treatments
Stock solution of the extract (40 mg / mL) was prepared by mixing 8.0 g of the
extract and 200 mL distilled water in a 250 mL Erlenmeyer flask. Different dilutions of the
extract were then prepared. For 10 mg / mL concentration, 13.75 mL of the stock solution
was mixed with 41.25 mL distilled water; for 20 mg / mL concentration, 27.50 mL stock
solution was mixed with 27.50 mL ditilled water; for 30 mg / mL concentration, 41.25 mL
stock solution was mixed with 13.75 mL distilled water; and for 40 mg / mL concentration,
no dilutions were made.
For the positive control, 3 M of copper (I) chloride (cuprous chloride) was prepared.
Powdered copper (I) chloride was weighed using an electronic weighing balance and 13.36 g
of the powder was dissolved in 45 mL distilled water.
Mung Bean Assay
11

Mung beans or green-gram seeds weighing 854 mg were used in the experiment. Six
seeds were dropped into each well containing 15 mL of the treatments. These setups were
replicated 3 times. The plates were closed with the lid and were left at room temperature for
24 (T-0) and 48 hours (T-1) of imbibition.
After the exposure to the treatments, the seeds were blot dried using paper towels.
They were then weighed one at a time using an electronic weighing scale. The percent
imbibition inhibition was calculated using the formula (G. Satyanarayana Murthy, et. al,
2011):
% = (

) 100

Wherein,
= wet weight of beans exposed to distilled water
= wet weight of beans exposed to the extract
= wet weight of beans exposed to copper (I) chloride
For further morphometric analysis, the time of sprouting was extended to 168
hours (1 week). Seedling length and radicle length were measured and the percent growth
(seedling length and radicle length) retardation was calculated using the formula:
% ( ) = (

) 100

Wherein,
= length of seedling or radicle exposed to distilled water
= length of seedling or radicle exposed to the extract

12

RESULTS AND DISCUSSION

Water hyacinth is an invasive plant with useful biological components. It has been
found out to contain alkaloids, ellagic acid, phenols, steroids, tannins, triterpinoids and
saponins. The work presented herein focused on the mitodepressive activity of water
hyacinth leaf extract through mung bean bioassay.
Table 1: Effect of water hyacinth leaf extract on seedling and shoot growth of Vigna radiata at
168 h treatment

Radicle Length
Seedling Length
Mean (mm)
Reduction Mean
Mean (mm)
Reduction Mean
SEM (mm)
(%) SEM (%)
SEM (mm)
(%) SEM (%)
ab
ab
10 mg / mL
3.44 0.12
48.44 0.06
8.17 0.23
19.1 0.10
20 mg / mL
3.11 0.11ab
53.38 0.06
7.61 0.27 ab
24.67 0.14
30 mg / mL
2.61 0.22ab
60.94 0.69
7.28 0.24 ab
27.92 0.03
ab
ab
40 mg / mL
3.33 0.20
50.12 0.48
8.22 0.17
18.59 0.30
Distilled Water
6.67 0.2
00.00
10.01 0.32
00.00
a
b
Significant difference at p < 0.001, Significant difference at p < 0.05 with Students t- test
(compared with the negative control)
Concentration

12
10
8
6

Radicle Length
Seedling Length

4
2
0
10 mg / mL

20 mg / mL

30 mg / mL

40 mg / mL

Distilled Water

Figure 4: Effect of water hyacinth leaf extract on seedling and shoot growth (mm) of Vigna radiata at 168 h treatment

Table 1 summarizes the results for the effect of water hyacinth leaf extract on the
growth of radicle and seedling of mung bean after 1 week of exposure to the treatments.
Water hyacinth leaf extract (10 mg / mL, 20 mg / mL, 30 mg / mL and 40 mg / mL) did not
13

induce dose dependent reduction in both radicle and seedling length of mung bean seedlings
(Figure 1). The radicle growth retardation increased from 48.44% 0.06% to 60.94%
0.69% for the increased concentration of 10 mg / mL to 30 mg / mL; however, it decreases to
50.12% 0.48% for 40 mg / mL treatment. The seedling growth, on the other hand,
increased from 19.1% 0.10% to 27.92% 0.03% for the increased concentration of 10 mg /
mL to 30 mg / mL and decreased to 18.59% 0.30% for 40 mg / mL treatment. Using
Students t-test statistics, all the concentrations of water hyacinth leaf extract has been found
out to exhibit significant radicle and seedling length reduction (p<0.05 & p<0.001).
Growth retardation of radicle brought about by water hyacinth leaf extract could have
resulted from the interference of the transition from the metabolically quiescent stage to the
rapidly dividing stage where cells in G1 phase shift to G2 phase; thus, it could have inhibited
the cell division (G. Satyanarayana Murthy, T.P. Francis, C. Rajendra Singh, H.G. Nagendra
and Chandrashekhar Naik, 2011).
Table 2: Effect of the treatments on the mean weight and percent imbibition inhibition of Vigna
radiata at T-0 and T-1

T-0 (24 hours)

T-1 (48 hours)
Mean Weight
Percent
Mean Weight
Percent
(mg) SEM
Inhibition (%)
(mg) SEM
Inhibition (%)
(mg)
SEM (%)
(mg)
SEM (%)
ab
ab
10 mg / mL
156.6 4.5
49.34 0.08
178.6 5.0
38.19 1.14
20 mg / mL
149.1 2.6ab
65.08 0.09
170.7 2.6ab
49.32 1.80
b
b
30 mg / mL
144.9 3.9
74.20 0.08
147.4 3.9
82.76 1.35
40 mg / mL
166.8 2.2ab
27.30 0.15
183.4 2.2ab
31.11 3.23
Cuprous chloride
132.7 2.4
100.00
135.3 2.4
100.00
ab
ab
Distilled water
179.8 5.3
00.00
205.3 5.3
00.00
a
b
Significant difference at p < 0.001, Significant difference at p < 0.05 with Students t- test
(compared with the positive control)
Concentration

14

The weight of mung bean seeds treated with water, cuprous chloride, and the different
concentrations of water hyacinth leaf extract and the percentage imbibition inhibition are
shown in Table 2. Calculation of the percentage imbibition inhibition, as suggested by G.
Satyanarayana Murthy, T.P. Francis, C. Rajendra Singh, H.G. Nagendra and Chandrashekhar
Naik (2011), clearly indicate the tendency of mitodepression in water hyacinth leaf extract.
This was due to the significant decrease (p<0.005 & p<0.001) of the weight of mung bean
seeds treated with the extract as compared to those treated with cuprous chloride, a known
mitotic-inhibitor.
The percentage imbibitions inhibition of the extracts are comparable to those of the
synthetic and herbal anticancer drugs as reported by G. Satyanarayana Murthy, T.P. Francis,
C. Rajendra Singh, H.G. Nagendra and Chandrashekhar Naik, 2011. Table 3 shows the
percentage imbibition inhibition of these drugs using mung bean bioassay.
Table 3: Percentage imbibition inhibition of some synthetic and plant extract anticancer drugs

Anticancer drugs
(synthetic)
Dacarbazine

% Imbibition
inhibition
25 %

Anticancer drugs
(plant extract)
Grass

% Imbibition
inhibition
42 %

Cyclophosphamide

36%

Teakwood

42 %

Leucovorin

43 %

Acacia

56 %

Flurouracil

43 %

Champaka

63 %

Vincristine

99 %

Bouganvilla

84 %

The results, therefore, indicate that water hyacinth leaf extract can cause mitotic
inhibition when tested through mung bean bioassay. The claim is consistent with the fact that
water hyacinth leaves contain phytochemicals such as alkaloids, ellagic acid, phenols,

15

steroids, tannins, triterpinoids and saponins which may interact with the mitotic apparatus (K.
Vasu, et al., 2009).

16

SUMMARY AND CONCLUSION

Significant results were attained for the mitodeppressive effect of methanolic extract
of water hyacinth leaves using mung bean bioassay. The capability on increasing the
percentage of imbibition inhibition was observed in the 10, 20, 30 and 40 mg of extract per 1
mL of distilled water. The trend decreased on the percentage of imbibition inhibition on the
40 mg of extract per 1 mL of distilled water. These results were based after 24 and 48 hours
of exposure with the same results in the two time intervals. The percentage of imbibition
inhibition achieved is comparable with commercialized and herbal anticancer drugs as
reported by G. Satyanarayana Murthy, T.P. Francis, C. Rajendra Singh, H.G. Nagendra and
Chandrashekhar Naik, 2011.
Also, the water hyacinth leaf extract (10 mg / mL, 20 mg / mL, 30 mg / mL and 40
mg / mL) did not show dose dependency in both radicle and seedling length of mung bean
seedlings after 168 hours of exposure. Growth retardation of radicle brought about by water
hyacinth leaf extract could have resulted from the interference of the transition from the
metabolically inactive stage to the rapidly dividing stage.
The results clearly illustrate that water hyacinth leaf extract can mitodepression on
mung beans.

17

RECOMMENDATIONS
The researchers used Mung Bean Bioassay to screen the antimitotic potential of water
hyacinth leaf extract. The assay was used because of its inexpensiveness and availability. In
the study, the researchers used the seeds that were available in the market and so, the results
gathered from the experiment have high standard deviation. It is therefore suggested to use
genetically cross-bred mung bean seeds in order to minimize the extraneous variables. By
using cross-bred seeds, it would be ensured that the subjects in the study are normal and
uniform.
The researchers were not able to examine then root length, which is one of the
morphometric parameters used in analyzing antimitotic drugs. In 168 hours of sprouting, no
roots have grown. This is maybe because the researchers did not place filter paper on the
wells and the time of exposure is not yet enough. It is therefore suggested to use filter paper
in mung bean sprouting and to extend the time of exposure to the treatments.
Lastly, it would be better to use commercially available anticancer drugs such as
methotrexate and cyclophosphamide as a positive control instead of using copper (I)
chloride. Through this, more accurate comparison to the experimental treatment can be
established.

18

BIBLIOGRAPHY
G. Satyanarayana Murthy, T.P. Francis, C. Rajendra Singh, H.G. Nagendra and
Chandrashekhar Naik, (2011). An assay for screening anti-mitotic activity of herbal
extracts.
Retrieved
December
15,
2013
from
http://webcache.googleusercontent.com/search?q=cache:xurC2ZS6NOEJ:www.curre
ntscience.ac.in/Volumes/100/09/1399.pdf+&cd=1&hl=en&ct=clnk
Gascoigne, K. & Taylor, S. (2009). How do anti-mitotic drugs kill cancer cells? Journal of
Cell Science 122, 2579-2585. doi: 10.1242/jcs.039719
Hernandez C.L., Villaseor I., Joseph E. and Tolliday N. (2008). Isolation and evaluation of
antimitotic activity of phenolic compounds from Pouteria campechiana Behni.
Retrieved
January
26,
2014
from
http://philjournalsci.dost.gov.ph/vol137no1/pdfs/Isolation%20and%20evaluation%20
of%20antimitotic%20activity%20of%20phenolic%20compounds.pdf
Kandukuri Vasu, Jakku Vinayasagar Goud, Aruri Suryam and M. A. Singara Charya (2009).
Biomolecular and phytochemical analyses of three aquatic angiosperms. Retrieved
January
26,
2014
from
http://www.academicjournals.org/article/article1380277547_kandukuri%20et%20al.p
df
Kehr

R.W. (2013).
Ellagic acid. Retrieved
http://www.cancertutor.com/ellagicacid/

January

26,

2013

from

Luciana Dobjanschi, Mariana Muresan, Angela Atonescu, Mihaela Zdrinca, Ildiko Szabo,
Mircea Tamas (2008). Antimitotic activity of saponins obtained from Anagalis
arvensis.

Retrieved

January

26,

2014

from

http://www.studiauniversitatis.ro/v15/pdf/18-2008/SU08Dobjanschi2.pdf
Mandal, A. (2013). What is cancer? Retrieved June 30, 2013 from http://www.newsmedical.net/health/What-is-Cancer.aspx
MediLexicon International Ltd. (n.d.). What is cancer? What causes cancer? Retrieved June
30, 2013 from http://www.medicalnewstoday.com/info/cancer-oncology/
PAN

Germany. (n.d.). Water hyacinth. Retrieved June 30, 2013

http://www.oisat.org/pests/weeds/broad_leaf_weeds/water_hyacinth.html

from

19

Patil S., Narayanan S., Eibl G. and Jolly C.L. (2004). Evaluation of antimitotic activity of
Rotula aquatica (Lour): a traditional herb used in treatment of cancer. Retrieved
January 26, 2013 from http://www.ncbi.nlm.nih.gov/pubmed/15462182
Penfound, W.T. & Earle, T.T. (1948). The biology of the water hyacinth. Ecological
Monographs, 18, 447-472.
Rajan D., Blesson J., Chandran S., Thampatty A.R. and Veena P.V. (2013). Detailed analysis
on phytochemicals, antioxidants, antimicrobial activity of Eichhornia crassipes.
Retrieved January 26, 2013 from
http://theglobaljournals.com/ijsr/file.php?val=NDQw
Sanjib Bhattacharya and Pallab K. Haldar (2012). Evaluation of antimitotic and genotoxic
effects of the triterpenoid enriched extract from Trichosanthes dioica root. Retrieved
January 26, 2014 from http://www.idosi.org/aejts/4(1)12/5.pdf
Thenmozhi, A., Nagalakshmi, A., Rao, M. (2011). Study of cytotoxic and antimitotic
activities of Solanum nigrum by using Allium cepa root tip assay and cancer chemo
preventive activity using MCF-7- human mammary gland breast adenocarcinoma cell
lines. International Journal of Science and Technology, 1, 2250 141X. Retrieved
June 30, 2013 from http://ijst.co.in/papers/vol1issue2/ijst_121104.pdf
Vaidya, G. & Baral, B. (n.d.). Biological and chemical assessment of water hyacinth
(Eichhorna Crassipes (Marts.) Solms.) Ofphewa, Lake Nepal. Retrieved August 7, 2013 from
http://www.academia.edu/1088390/BIOLOGICAL_AND_CHEMICAL_ASSESSME
NT_OF_WATER_HYACINTH_Crassipes_
Wersal, R. & Madsen, J. (2012). A review of the global status of aquatic plants. Aquatic
plants
their
uses
and
risks
Retrieved
June
30,
2013
from
http://www.gri.msstate.edu/publications/docs/2012/03/10109FAO_IPPC_Aquatic_Pl
ants_2012x.pdf
World Cancer Research Fund International. (n.d.). Cancer statistics. Retrieved June 30, 2013
from http://www.wcrf.org/cancer_statistics/world_cancer_statistics.php

20

APPENDIX A
RAW DATA
I.

Weight of Seed (mg)

T-0 (24 Hours)

10 mg / mL
Extract
88.1
137.2
154.2
154.6
155.5
156.3
157.1
157.8
160.9
161.8
162.2
163.8
165.3
166.2
166.3
166.4
166.5
178.2

20 mg / mL
Extract
127.8
128.6
132.3
136.1
145.6
146.4
146.4
147.1
151.8
155.9
156.0
156.9
157.1
157.4
157.8
158.3
161.5
161.5

30 mg / mL
Extract
94.2
123.7
136.2
136.2
138.0
138.6
140.1
140.3
153.0
153.7
154.4
154.6
156.1
156.2
156.6
158.2
159.2
159.5

10 mg / mL
Extract
98.1
169.2
176.3
176.7
177.6
178.3
179.2
179.9
183.0
183.9
184.3
185.9
187.4
188.3
188.4
188.5
189.6
200.3

20 mg / mL
Extract
149.4
150.2
153.9
157.7
167.2
168.0
168.0
168.7
173.4
177.5
177.6
178.5
178.7
179.0
179.4
179.9
183.1
183.1

30 mg / mL
Extract
96.7
126.2
138.7
138.7
140.5
141.1
142.6
142.8
155.5
156.2
156.9
157.1
158.6
158.7
159.1
160.7
161.7
162.0

40 mg / mL
Extract
152.7
152.9
156.1
158.4
161.4
161.5
162.8
164.3
164.5
167.8
169.8
171.8
172.6
172.8
172.9
173.3
183.6
183.9

Copper (I)
chloride
107.5
120.2
122.9
124.4
125.6
128.0
128.9
130.6
132.1
136.2
136.2
136.7
137.8
141.8
142.5
144.7
144.9
148.1

Distilled
Water
103.8
166.6
166.8
167.3
169.8
173.2
173.9
176.4
188.3
188.3
192.0
192.2
193.1
193.6
194.3
194.6
199.8
202.42

Copper (I)
chloride
110.1
122.8
125.5
127.0
128.2
130.6
131.5
133.2
134.7
138.8
138.8
139.3
140.4
144.4
145.1
147.3
147.5
150.7

Distilled
Water
129.3
192.1
192.3
192.8
195.3
198.7
199.4
201.9
213.8
213.8
217.5
217.7
218.6
219.1
219.8
220.1
225.3
277.9

T-1 (48 hours)

40 mg / mL
Extract
169.3
169.5
172.7
175.0
178.0
178.1
179.4
180.9
181.1
184.4
186.4
188.4
189.2
189.4
189.5
189.9
200.2
200.5

21

II.

Length of Seeds (mm)

10 mg / mL
Extract
6.0
6.0
7.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0
III.

20 mg / mL
Extract
6.0
6.0
6.0
6.0
7.0
7.0
7.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
9.0
9.0
9.0
9.0

30 mg / mL
Extract
5.0
6.0
6.0
6.0
7.0
7.0
7.0
7.0
7.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
9.0

40 mg / mL
Extract
7.0
7.0
7.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
9.0
9.0
9.0
9.0
9.0
9.0
9.0

Distilled Water

30 mg / mL
Extract
0
2.0
2.0
2.0
2.0
2.0
2.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
4.0
4.0
4.0

40 mg / mL
Extract
2.0
2.0
2.0
2.0
3.0
3.0
3.0
3.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0

Distilled Water

8.0
8.0
8.0
8.0
9.0
10.0
10.0
10.0
10.0
10.0
11.0
11.0
11.0
11.0
11.0
11.0
12.0
12.0

Length of Radicles (mm)

10 mg / mL
Extract
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0

20 mg / mL
Extract
2.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
4.0
4.0
4.0

5.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
7.0
7.0
7.0
7.0
7.0
8.0
8.0
8.0
8.0
22

APPENDIX B
STATISTICAL ANALYSIS
I.

II.

Weight of Mung Bean (24 hours)

10 mg /
mL
Extract

20 mg /
mL
Extract

30 mg /
mL
Extract

40 mg /
mL
Extract

Copper
(I)
chloride

Distilled
Water

Mean

178.6 mg

170.7 mg

147.4 mg

183.4
mg

135.3 mg

205.3 mg

Median

183.5 mg

175.5 mg

155.9 mg

182.8
mg

136.8 mg

213.8 mg

Mode

ALL

168.0
mg,
183.1 mg

138.7 mg

ALL

138.8 mg

213.8 mg

SD

21.2428

11.1638

16.4759

9.1413

10.3562

22.5178

Variance

451.2558

124.6296

271.4553

83.5637

107.2515

507.0529

SEM

5.0 mg

2.6 mg

3.9 mg

2.2 mg

2.4 mg

5.3 mg

Copper
(I)
chloride
132.7 mg

Distilled
Water
179.8 mg

134.1 mg

188.3 mg

136.2 mg

188.3 mg

10.3562
107.2515
2.4 mg

22.5178
507.0529
5.3 mg

Weight of Mung Bean (48 hours)

Mean

10 mg /
mL
Extract
156.6 mg

20 mg /
mL
Extract
141.1 mg

30 mg /
mL
Extract
144.9 mg

Median

161.4 mg

153.9 mg

153.4 mg

Mode

ALL

136.2 mg

SD
Variance
SEM

18.9932
360.7430
5.0 mg

146.4
mg,
161.5 mg
11.1638
124.6296
2.6 mg

40 mg /
mL
Extract
166.8
mg
166.2
mg
ALL

16.4759
271.4553
3.9 mg

9.1413
83.5637
2.2 mg

23

III.

Radicle Length (168 hours)

Mean
Median
Mode
SD
Variance
SEM

IV.

VI.

20 mg / mL
Extract
3.11 mm
3.00 mm
3.00 mm
0.4714
0.2222
0.11 mm

30 mg / mL
Extract
2.61 mm
3.00 mm
3.00 mm
0.9164
0.8398
0.22 mm

40 mg / mL
Extract
3.33 mm
4.00 mm
4.00 mm
0.8401
0.7058
0.20 mm

Distilled
Water
6.67 mm
6.50 mm
6.00 mm
0.9074
0.8235
0.21 mm

30 mg / mL
Extract
7.28 mm
7.50 mm
8.00 mm
1.0178
1.0359
0.24 mm

40 mg / mL
Extract
8.22 mm
8.00 mm
8.00 mm
0.7320
0.5359
0.17 mm

Distilled
Water
10.1 mm
10.00 mm
11.00 mm
1.3492
1.8203
0.32 mm

Seedling Length (168 hours)

Mean
Median
Mode
SD
Variance
SEM
V.

10 mg / mL
Extract
3.40 mm
3.00 mm
3.00 mm
0.5113
0.2614
0.12 mm

10 mg / mL
Extract
8.17 mm
8.00 mm
9.00 mm
0.9851
0.9706
0.23 mm

20 mg / mL
Extract
7.61 mm
8.00 mm
8.00 mm
1.0921
1.1928
0.27 mm

Students t-test Statistic (Seedling Length Compared with the Negative Control)
Concentration

t value

10 mg/mL
20 mg/mL
30 mg/mL
40 mg/mL

4.9015
6.0860
7.0792
5.1963

p=0.05
(p<2.0322)
Success
Success
Success
Success

p=0.001
(p<3.6008)
Success
Success
Success
Success

Students t-test Statistic (Radicle Length Compared with the Negative Control)
Concentration

t value

10 mg/mL
20 mg/mL
30 mg/mL
40 mg/mL

13.3201
13.5675
13.3566
11.4593

p=0.05
(p<2.0322)
Success
Success
Success
Success

p=0.001
(p<3.6008)
Success
Success
Success
Success

S
24

VII.

VIII.

Students t-test Statistic (Imbibition of Extract Compared with CuCl2 at T-0)

Concentration

t value

10 mg/mL
20 mg/mL
30 mg/mL
40 mg/mL

4.6774
4.5721
2.6598
10.4760

p=0.05
(p<2.0322)
Yes
Yes
Yes
Yes

p=0.001
(p<3.6008)
Yes
Yes
No
Yes

Students t-test Statistic (Imbibition of Extract Compared with CuCl2 at T-1)

Concentration

t value

10 mg/mL
20 mg/mL
30 mg/mL
40 mg/mL

7.7694
9.8660
2.6392
14.7766

p=0.05
(p<2.0322)
Yes
Yes
Yes
Yes

p=0.001
(p<3.6008)
Yes
Yes
No
Yes

25

APPENDIX C
DOCUMENTATION

Soxhlet Extraction

Set-Ups
26

Weighing of the mung bean

seeds

Measuring of the mung bean seeds

27