CYTOTOXIC AND GENOTOXIC POTENTIALS OF THE MONEY TREE

(Pachira aquatica) STEM AND LEAF EXTRACTS

Jane Nicole N. Catacutan

Melissa C. Gaudario
Maries Ann R. Silvestre
Researchers

Mary Lorraine F. Lorido

Research Adviser

Jordan Ferdin A. Halili

Rich Milton R. Dulay
Research Consultants

Juan R. Liwag Memorial High School

Bayanihan, City of Gapan, Nueva Ecija
 CYTOTOXIC AND GENOTOXIC POTENTIALS OF MONEY TREE
(Pachira aquatica) STEM AND LEAF EXTRACTS

Jane Nicole N. Catacutan

Melissa C. Gaudario
Maries Ann R. Silvestre

ABSTRACT

The discovery and development of chemotherapeutic drugs is a continuous worldwide demand.

This study was designed as an anticancer prescreening to evaluate the cytotoxic and genotoxic potentials
of leaf and stem extracts of Money tree (Pachira aquatica), one of the unexplored plant species.

Bioactivity of MT extracts was initially assessed using brine shrimp lethality assay (BSLA). Plant
and animal models of cell proliferation were used to investigate cytostatic and cytocidal effects. Onion
root tip chromosomal aberration assay (ORTCAA) was conducted to examine antimitotic and genotoxic
activities. Embryotoxicity and teratogenicity were determined in zebrafish developmental toxicity assay
(ZDTA).

Using BSLA, MT stem and leaf extracts had estimated LC50 values of 104.75 and 121.69 μg/ml
respectively which indicated that both extracts were bioactive/toxic. ORTCAA revealed that all stem
extract concentration reduced mitotic indices which were comparable to Maleic hydrazide (positive
control) while all leaf extract concentrations induced mitotic block at prophase/metaphase boundary.
Prominent chromosomal aberrations observed were bridges and stickiness suggesting genotoxicity of
extracts. ZDTA showed 100% embryonic death at 20, 100 and 200 μg/ml of both extracts after 12-hour
post-treatment application. Moreover, cytological abnormalities in onion cells and early zebrafish
embryonic death implied the activation of apoptosis.

Though the results cannot be confirmed generally whether the extracts could genotoxic or
cytotoxic or both, the extracts have promising cytostatic (inhibition of growth, division and
differentiation) and cytocidal (lethal) effects, important fates of an anticancer drug and is therefore a
potential source of a nature-based chemotherapeutic compound.
 TABLE OF CONTENTS
Page
Title Page .....................................................................................................................................i
Abstract ........................................................................................................................................ii
Table of Contents .........................................................................................................................iii
List of Tables ...............................................................................................................................v
List of Images ..............................................................................................................................vi
List of Figures .............................................................................................................................viii
INTRODUCTION .......................................................................................................................1
METHODOLOGY ......................................................................................................................3
Collection and Identification of Plant Samples ...............................................................4
Preparation of Crude Ethanolic Extracts and Phytochemical Analysis ...........................4
Preparation of Treatments ................................................................................................4
Brine Shrimp Lethality Assay ..........................................................................................5
Onion Root Tip Chromosomal Aberration Assay ...........................................................6
Zebrafish Developmental Toxicity Assay .......................................................................7
Data Analysis ...................................................................................................................9
Waste Disposal.................................................................................................................9
RESULTS AND DISCUSSION ..................................................................................................10
Plant Authentication and Scientific Classification ..........................................................10
Phytochemical Analysis ...................................................................................................10
Brine Shrimp Lethality Assay ..........................................................................................11
Onion Root Tip Chromosomal Aberration Assay ...........................................................13
Zebrafish Developmental Toxicity Assay .......................................................................22
CONCLUSIONS .........................................................................................................................28
RECOMMEDATIONS ................................................................................................................30
REFERENCES ............................................................................................................................31
ACKNOWLEDGEMENT ...........................................................................................................40
 INTRODUCTION

According to the American Cancer Society (2015), 1 out of 7 deaths is caused by cancer.

This global health burden is among the leading causes of mortality, accounted for 8.2 million

deaths in 2012 (WHO, 2015). In the Philippines, 98,200 individuals were affected by cancer each

year wherein more than half of these patients died in 2014 (Asian Cancer Institute, 2015). These

statistics at an alarming pace increased the worldwide demand to cure and prevent this prevalent

disease.

Despite of the advancements in the development of treatments for cancer which include

chemotherapy, radiation and targeted therapy to destroy, stop rapid proliferation and/or delay the

growth of these aggressive cells (US National Cancer Institute, 2007) until now, there is no

existing ideal anticancer mechanism which could only kill cancer cells and spare healthy cells

(Amara, 2013). Moreover, chemically-synthesized drugs result to a wide range of detrimental

effects and/or even total destruction of non-targeted sites in the human body (Seideil et al. 2012).

The search for chemotherapeutic agents with tolerable secondary effects at its greatest cytotoxic

action had been a continuous demand in the field of biomedical and health sciences (Coseri,

2009).

Plants and its secondary metabolites had been an abundant source of anticancer agents

(Newman, 2008) since these nature-based compounds exhibit a wide range of cytotoxic activities

and promising selective bioactivity (Cragg and Newman, 2005). In this study, the researchers

investigated the cytotoxic activity of money tree scientifically known as Pachira aquatica which

is considered as one of the unexplored plant species (Lawal et. al 2015) since limited scientific

study of its bioactivity only includes the antifungal activity of its root (Shibatani et al. 1999).
Other countries traditionally use MT for bacterial infections, gastric problems, diabetes, skin

irritations and blood purification (Pietsch et al. 2009).

Generally, this study aimed to evaluate the cytotoxic activity of by investigating

cytostatic (stopping cell growth, division, function and differentiation) and cytocidal (killing

cells) mechanisms in plant and animal models as an anticancer prescreening. Antimitotic,

genotoxic, embryotoxic and teratogenic activities were assessed to determine the mode and site

of cytotoxic effects of the phytocompounds present in MT extract which could be a significant

baseline data in the extensive efforts of developing novel nature-based chemotherapeutic drugs.
 METHODOLOGY

Figure 1. Flow chart of Materials and Methods

 MATERIALS AND METHODS

I. Collection and Identification of Plant Samples

The money tree (MT) (Pachira aquatica) leaves and stems were collected from San Nicolas,

Gapan City, Nueva Ecija and were brought to Botany Division, National Museum, Ermita,

Manila for identification and authentication.

II. Preparation of Crude Ethanol Extracts and Phytochemical analysis

Five kilograms each of fresh leaves and stem of MT (Pachira aquatica) were collected

from San Nicolas, Gapan City and brought to Department of Science and Technology (DOST),

Maimpis, City of San Fernando, Pampanga for ethanol extraction. Leaves and stems were

washed and air-dried. The plant samples were then powdered and macerated with 95% EtOH

(1:5 w/v) at room temperature for 72 hours. The extracts were filtered and further evaporated

under reduced pressure using a rotary evaporator at 55 °C (Souza et al. 2014).

Phytochemical analysis of the crude extracts was also done at the Industrial Technology

Development Institute (ITDI), Department of Science and Technology (DOST), Maimpis, City

of San Fernando, Pampanga.

III. Preparation of Treatments

For the Brine Shrimp Lethality assay various dilutions of each of crude extracts of MFT

leaves and stems were prepared giving final concentrations ranging from 0 to 100 μg/ml,

following the modified dilution procedure of McLaughlin et al. (1991). Negative control

(artificial sea water with 1% DMSO) and positive control (potassium dichromate) were also

included. There were 6 replicates with 2 trials each including the test controls giving a total of 8

treatments.
 5
For the Onion Root Tip Chromosomal Aberration Assay, 100 μg each of MFT leaf and stem

extracts were dissolved in 10 ml of distilled water with 1 ml DMSO to give varying

concentrations from 100 to 1000 μg/ml. A total of 4 treatments were prepared each in 3

replicates. Positive (maleic hydrazide) and negative (distilled water with 1% DMSO) were also

included.

In the Zebrafish developmental toxicity assay, 20 µg leaf and stem extracts were added to 20

ml embryo medium. The final concentrations were 2, 10, 20, 100 and 200 μg/ml. Embryo

medium was used as the negative control. A total of 6 treatments were prepared each in 3

replicates.

IV. Brine Shrimp Lethality Assay (BSLA)

Three milligrams of brine shrimp (Artemia salina) eggs were hatched in a beaker

placed with 400 mL of artificial sea water with 3.8% sodium chloride solution under constant

aeration and illumination of fluorescent lamp for 48 hours.

6
After hatching, 10-15 brine shrimps were transferred in a 24-well plate containing

different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/ml) each of MFT leaves and stem

extracts. Potassium dichromate and 1% DMSO each dissolved in artificial seawater served as the

positive and negative controls, respectively. The absence of the action of brine shrimps within 30

seconds was considered to determine the mortality of nauplii (Middleton et al. 2005) and if they

were lying at the bottom of the container (Tawaha, 2005). After the 24 hours, the dead Artemia

salina were counted manually wherein the percentage mortality (%M) was calculated by

dividing the number of dead brine shrimps by the total number of brine shrimps, then multiplied

by 100. Abbott’s mortality formula was used to correct the percentage lethality of brine shrimps

to ensure the bioactivity of extracts (Meyer et al. 1982).

 Abbott’s mortality formula

% mortality due to treatment = _y – x x (100)

100-x

wherein: x = no. of dead brine shrimps in the control

y = no. of dead nauplii in the treatment vials

The median lethal concentration (LC50) was computed using the generated equation of

the line in the Trend Line Fit Regression analysis in Microsoft Excel 2013. Median lethal

concentration with a value less than 1000 μg/ml was considered toxic while LC 50 value greater

than 1000 μg/ml was non-toxic (Meyer et al.1982).

V. Onion Root Tip Chromosomal Aberration Assay (ORTCAA)

Forty onions (Allium cepa) of common variety and equal sizes were obtained commercially

from a local market at Muñoz, Quezon City. A scalpel was used in peeling the outer scales of the

bulbs and old roots to expose the root primordia. Onions were further examined for uniformity

and condition wherein 18 bulbs were randomly selected. Stem and leaf extracts of Pachira

aquatica were prepared in different concentrations at 100, 250, 500 and 1000 µg/ml including

the positive (Maleic hydrazide) and negative control (distilled water with 1% DMSO) on onion

bulbs with three replicates for 48 hours.

7
Onion (Allium cepa) root tips were then cut and fixed with Farmer’s fluid (1-part glacial

acetic acid and 3 parts of ethanol) in plastic micro-tubes and placed in the refrigerator after 24

hours. Root tips were then cut to 1-2 mm, hydrolyzed with a drop of 1N HCL (hydrochloric acid)

for 3 minutes, dried with a tissue paper and macerated using scalpel. Aceto orcein (2%) was used

to stain the onion cells for 3 minutes, then passed the glass slide over the flame of alcohol lamp

twice to avoid the stain to boil. Cover slip was used to squash the cells and remove excess stain

wherein it was sealed with a natural nail polish. The slide samples were studied using Zeiss
compound light microscope under 10X objective then switched to 40X objective to take closer

observation in the morphological structure and mitotic phase of cells. This bioassay procedure

with some modifications was used according to El-Shahaby et al. (2003).

The mitotic index, mitotic phase indices and percentage of aberrant cells were calculated

using the formula below (Fiskesjo, 1985):

Number of dividing cells

Mitotic Index = --------------------------------- x 100
Total number of cells

Number of dividing cells (P/M/A/T)

Mitotic Phase Index = -------------------------------------------------- x 100
Total number of dividing cells

Number of aberrant cells

% of Aberrant cells = --------------------------------- x 100
Total number of cells

In addition, chromosomal aberrations such as lagging, loss and bridging were identified.

Investigation of other structural abnormalities in the nucleus and cytoplasm were also done.

VI. Zebrafish Developmental Toxicity Assay (ZDTA)

Zebrafish (Danio rerio) was obtained at a market place in San Jose, Nueva Ecija. The ZDTA

was conducted in the Department of Biological Science, College of Arts and Sciences, Central

Luzon State University, Science City of Muñoz, Nueva Ecija.

8
The protocol of this study was based after Nagel (2002). Dechlorinated water with

oxygen saturation in a glass aquarium was used for the spawning of zebrafish at 1:2 ratio

between mature females to males. Adult (10-12 months of age) zebrafishes were placed in a

plastic mesh and were submerged in the aquarium for localization. It was covered with black

plastic bag for 12 hours to induce spawning.

 After incubation in the dark, eggs were exposed to a well-lighted (300-320 lux) condition

for another 12 hours wherein fertilization typically occurred after 30 minutes. The fertilized eggs

were siphoned out of the aquarium using a hose and aspirator bulb to isolate it from the water.

Embryos were rinsed thrice using distilled water and were placed in a watch glass with an

embryo medium and observed under the compound microscope to examine its uniformity and

condition.

Four selected embryos were transferred to 24-well plates with 3 ml of each concentration

of the stem and leaf extracts (2, 10, 20, 100, 200 µg/ml and embryo water as negative control)

and incubated at 26°C ± 1°C. Observations were made after 12, 24, 36 and 48 hours of

incubation using a compound microscope with a High Power Objective (HPO) at 40X

magnification. Hatchability, malformation and mortality rates were observed and recorded. The

experimentation was performed under the protocol of national guidelines for animal welfare by

Nagel (2002).

VII. Data Analysis

Comparison between groups were made by means of One-Way Analysis of Variance with

Duncan Multiple Range Test with the aid of SPSS version 22.0. Differences with p<0.01

between experimental and control groups were considered.

VIII. Waste Disposal

Chemicals and solvents used were disposed in appropriate organic and inorganic waste

containers. Solid wastes were collected in a waste container and disposed. (Laboratory Health,

Safety and Environment Management System - University of the Philippines Diliman).

 Brine shrimps exposed in different treatments were treated with 10% bleach solution for 24

hours before its disposal in a black plastic bag (National Science Foundation, 2013). Zebrafish

embryos were subjected to euthanasia by means of submersion in ice water for at least 20

minutes prior to disposal (National Institutes of Health, 2013).

 RESULTS AND DISCUSSION

Plant Authentication and Scientific Classification

The money tree plant sample used in the study was identified and authenticated as

Pachira aquatica Aublet of Malvaceae family by the Botany Division, National Museum in

Ermita, Manila.

Phytochemical Analysis

A qualitative phytochemical analysis was carried out to identify the physiological active

components of MT stem and leaf extracts. The table below shows the result of screening and test

methods used to determine the presence of the bioactive compounds on the stem and leaf of

money tree (Pachira aquatica).

Table 1. Phytochemicals present in money tree (Pachira aquatica) stem and leaf extracts
TEST PARAMETER TEST METHOD LEAF STEM
Alkaloids Mayer/Meyer Test + +
Anthraquinones Borntrager Test - -
Glycosides Keller-Killiani Test + +
Bate-Smith and Metcalf
Flavonoids - -
Test
Tannins Ferric chloride Test + +
Saponins Froth Test - -
Legend: + presence; - absence

The ethanolic extract of both stem and leaf exhibited the presence of the same

compounds namely alkaloids, glycosides, and tannins (condensed). Thus, the resulting

mechanisms of antimitotic, genotoxic, embryotoxic and teratogenic activities in this study could

be attributed to the phytochemical constituents present in Pachira aquatica stem and leaf

extracts.

Several alkaloids such as vinca alkaloids were significant chemical compounds included

in proven chemotherapy drugs (Moudi et al. 2013). These active components exhibited marked
alkylating properties or ability to combine with deoxyribonucleic acid and inhibit the progression

of cancer cells. Moreover, according to studies alkaloids have the capability to target signals, ion

channels, receptors, DNA, proteins and vital cell organelles in animal cells (Fattorusso and

Taglialatela-Scafati, 2007). Futhermore, tannins were prominent for its anticancer properties by

means of hindering enzyme production which is a vital process for cancer cells. Particularly,

Nandakumar et al. (2008) documented the promising anticancer and antioxidant activities of

condensed tannins. On the other hand, glycosides do not only induce the death of cancer cells but

it also triggers the immune system to kill other cancer cells (Menger et al. 2012).

Brine Shrimp Lethality Assay (BSLA)

Brine shrimp (Artemia salina) lethality assay was used to the examine the bioactivity of

the money tree leaf and stem compounds (Lieberman, 1999) This arthropod assay was

internationally recognized as a prescreening for antitumor activity (Meyer et al. 1982) and its

prominent positive correlation with human cancer line tests (McLaughlin and Rogers 1998;

Carballo et al. 2002; Naidu et al. 2014).

Bioactivity was determined by the

lethality of brine shrimp exposed to the

extracts for 24 hours. Figure 2 shows the

effect of MFT stem extract on the mortality

of brine shrimp nauplii under the

experimental conditions used.

12
Figure 2. The effect of Pachira aquatica stem
The results showed that the ethanolic extract
extractof
on Pachira aquatica
the lethality stem salina
of Artemia was most active
nauplii

against Artemia salina nauplii at 25 µg/ml (53.82%) and lowest at 6.25 µg/ml (41.15%). The

bioactivity of the phytocompounds showed a gradual increase with concentration resulting to the
mean percentage mortalities of 42.79% (3.125 µg/ml), 42.97% (6.25 µg/ml), 46.89% (50 µg/ml)

and 48.17% (100 µg/ml).

Similarly, MT leaf extract

recorded its maximum nauplii mortality at

25 µg/ml (80.86%) while lowest at 100

µg/ml (49.79%). (See Figure 3). Despite

of the decreasing trend, other

concentrations killed sufficient number of

Figure 3. The effect of Pachira aquatica leaf
brine shrimp nauplii at 12.5 µg/ml
extract on the mortality of brine shrimp nauplii
(77.61%), 6.25 µg/ml (64.47%), 50 µg/ml (61.26%) and 3.125 µg/ml (54.48%). In a cytotoxic

analysis by Pourfraidon and Sharma (2009) using 19 plants with prominent anticancer properties,

42% of the extracts exhibited 61-100% brine shrimp inhibition, 32% of the investigated plants

killed 31-60% of the nauplii and 26% had weaker cytotoxic activity resulting with 0-30%

Artemia salina lethality after 24 hours. The MT extracts therefore have potent cytotoxic behavior

against brine shrimps.

The median lethal concentration (LC50) of stem and leaf extracts was estimated using

trend line fit regression analysis wherein the calculated values were 104.75 µg/ml and 121.69

µg/ml, respectively. According to Meyer’s Toxicity Index (1982), LC 50<1000 µg/ml was

considered toxic while LC50>1000 µg/ml was non-toxic. The results indicate that both extracts

contain significant bioactive compounds that are responsible for toxicological activity (Nguta et

al. 2012). Moreover, substances with LC50<200 µg/ml in BSLA could be further subjected to cell

culture to detect its antitumor activity (Romeo, 2012). However, despite that LC 50 values may
not provide the exact concentration of the extract that can kill 50% of Artemia salina, it could

provide a baseline data for further bioactivity analysis (Hamidi et al. 2014).

Meyer et al. (1982) investigated 12 physiologically active components, three of these:

podophyllotoxin, barberine chloride and strychnine sulfate which have LC50 values less than 100

µg/ml. The other compounds have LC50 values greater than 150 µg/ml. Moreover, Bagya et al.

(2011) tested Vincristine, a nature-based chemotherapeutic drug in brine shrimp assay wherein it

was recorded with a median lethal concentration value of 380 µg/ml. In this study, the computed

LC50 values of Pachira aquatica stem and leaf extracts were 104.75 µg/ml and 121.69 µg/ml

respectively which suggest that the plant could be an abundant source of bioactive compounds

and a candidate for further confirmatory testing of cytotoxic mechanisms that could be exhibited

in cell growth, function, division and differentiation (Goodman et al. 1980) and the ability to

activate its self-destruction machineries or cytocidal effect (Foster, 2015).

Onion Root Tip Chromosomal Aberration Assay (ORTCAA)

Onion (Allium cepa) root tip chromosomal aberration assay has been considered as a

reliable system since the division of onion cells, cancer cells and human somatic cells are

similar. ORTCAA was widely considered as a prescreening for anticancer/antimitotic

compounds (William and Omoh, 1996) since plant cells were noted to be 1000 times more

resistant to colchicine, a carcinogenic substance and well-known mitosis inhibitor (Kihlman,

1996). Furthermore, this assay was also proven for its practicality since one rootlet can show the

range of DNA damage in onions (Tedesco and Laughinghouse IV, 2012).

A. Antimitotic Activity

Mitotic index is an indicator of cell proliferation (Gadano et al. 2002) wherein its

significant reduction could be resulted by the inhibition of DNA/protein synthesis (Chandra et al.
2005), blockage of the G2 phase of cell cycle which causes delay in the cell cycle kinetics (Rojas

et al. 1993) and mitotic arrest (Kumar et al. 2006) that could eventually lead to apoptosis or cell

death (El-Ghamery et al. 2000).

15
Figure 4 shows the rate of mitosis

of Allium cepa root tip cells after 24 hours.

The mitotic indices of root meristems

treated with 100 µg/ml, 250 µg/ml, 500

µg/ml and 1000 µg/ml stem extract were

7.47%, 8.00%, 8.13% and 9.07%

respectively were not significantly different Figure 4. The mitotic indices of Allium cepa root
tip cells treated with MT stem extract
*Means having the same lower case letter data labels are
from each other. The mitotic activity of not significantly different (p<0.01)

onion root tip cells inhibited by the extract was statistically comparable to the positive control

(6.20%) and significantly lower compared to the negative control (21.87%). The results suggest

that the stem extract in varying concentrations have the same effects in inducing the

accumulation of interphase cells with Maleic hydrazide which is a known plant growth

depressant and comparable growth inhibitory activities with gamma irradiation (Gubb and

Mactavish, 2002).

Moreover, the division of meristem cells in all treatments were less than 50% of the

negative control which indicates the cytotoxic activity of the extract that inhibited dividing cells.

Furthermore, despite of not reducing MI up to 22% of the negative control to exhibit lethal

effects to an organism, the greatest action

of pre-prophase inhibition of 100 µg/ml

stem extract was found to be 34% of the

negative control, causing sublethal effects (Antosiewicz, 1990).

Figure 5 shows that there were higher percentage of cells in telophase for root meristem

cells treated with 100 µg/ml (78.57%) and 250 µg/ml (70%) stem extract. Prophase

accumulation was evidently observed in Allium cepa cells exposed with 500 µg/ml (87.70%) and

1000 µg/ml (89.71%) extract which were even higher than the positive control. There was also a

drastic decrease in the percentage of actively proliferating cells with 100, 500 and 1000 µg/ml

stem extract at metaphase and anaphase

Figure 5. The mitotic phase indices of Allium cepa
that was only ranging from 0.0-3.3%. root tip cells in MT stem extract

The mean percentage of onion root meristem cells at prophase in 500 µg/ml and 1000

µg/ml stem extract were significantly higher than the 2 control groups wherein according to

Prasad and Das (1977), high frequency of prophase cells was due to the prophase poisoning that

could trigger early mitotic arrest. Moreover, it could be possibly due to prolonged or arrest of

cells at prophase stage and spindle fiber formation disturbance (Deysson, 1968).

It can be noted in Figure 6 that

there is a significant decrease in the rate of

cell proliferation of onion cells treated

with 1000 µg/ml (8.47%) leaf extract

wherein this antimitotic action was

comparable with positive control.

Figure 6. The mitotic indices of Allium cepa root
Moreover, the pre-prophase inhibition of tip cells treated with Pachira aquatica leaf extract
*Means having the same lower case letter data labels are
the two highest concentration were not significantly different (p<0.01)

statistically the same and all of the MT leaf

extract levels were documented with

significantly lower mitotic indices than the negative control. In addition, the rate of blocking

onion root tip cells from entering mitosis was also increasing as concentration increases.

High frequency of prophase cells was evidently seen in all experimental treatments

wherein more than 45% of the dividing cells were accumulated in this stage. (See Figure 7). The

mean percentage of onion root meristem cells at prophase in all concentrations were significantly

higher than the negative control. It could be attributed to the action of the leaf extract to cause

interruptions in nuclear membrane disintegration due to the inhibitory effects carried from the

earlier phases of cell cycle (Wilson, 1965)

Figure 7. The mitotic phase indices of Allium cepa
delay or blockage in the formation of root tip cells in MT leaf extract

spindle fiber (Ilbas et al. 2011) and caused by prophase arrest (Njoku et al. 2015). In addition,

there was also a higher percentage of cells exposed with 1000 µg/ml at metaphase and anaphase

compared to the antimitotic action of MT stem extract which implies the action of leaf extract on

the mechanisms in the second and third stage of mitosis. All of the concentrations have

significantly lower percentage of cells at telophase.

Akinboro and Bakare (2007) investigated the effects of common medicinal plants on

onion cells wherein the percentage of dividing cells treated with the highest concentration of

Azadirachta indica, Morinda lucida, Cymbopogon citratus and Carica papaya ranges from 10-

18%. In an antitumor screening by Bagya et al. (2011) using Kaempferia galangal,

Clerodendrum viscosum, Jatropha curcas and Lens culinaris in onion root tip test, mitotic

indices at 50 µg/ml of the ethanolic extracts range from 20-60% while the 100 µg/ml resulted to

10-50%. Vincristine, a plant-derived anticancer drug was used as positive control which was

noted with 0-3% mitotic indices. Another traditional anticancer herb, Rotula aquatica was

documented with 12-19% mitotic indices in its aqueous, methotrexate and ethanol extracts (Patil
 et al. 2004). In this present study, the MI of the onion meristem cells treated with the lowest

concentration (100 µg/ml) of money tree stem and leaf extracts were 7% and 17% respectively

which were relatively lower than action of Clerodendrum viscosum, Jatropha curcus, Lens

culinaris, and Cymbopogan citratus since the greater the number of cells at interphase indicates

to a delay in the cell cycle kinetics (Udo et al. 2015) thus, exhibiting the inhibition of the mitotic

process.
18
Furthermore, similar effects of prophase accumulation in the dividing cells were also

seen in the studies using the leaf extract of Telfairia occidentalis, Vernonia amygdalina (Udo et

al. 2015), Aloe vera (Ilbas et al. 2011) and Azadirachta indica (Akaneme and Amaefule, 2012)

which is an indicator of prominent anti-spindular effects at the early stage of mitosis wherein this

direct interaction with spindle fibers was further validated in the telophase accumulation of cells

at lower concentrations of Pachira aquatica stem extract which was also a sign of spindle

disturbance and/or total breakdown (Udo et al. 2015). Results suggest the promising antimitotic

activity induced by MT extracts and its capability to build up cells at prophase which was also

the antimitotic action of Taxol and Taxotere, two clinically used anticancer drugs investigated

using ORTCAA (Agnieszka et al. 2008).

B. Genotoxic Activity

Exposure of the onion root tip

meristem cells in different concentrations

of Pachira aquatica stem extract resulted

chromosomal aberrations including

fragmentation (0.06%), chromosome

bridges (2.73%) and stickiness (4.2%).

Figure 8. The result of Allium cepa root tip cells
aberrations test in different concentrations of MT
stem extract
Legend: Orange bars, chromosomal aberrations; Blue bars,
cytological aberrations
 19
(See Figure 8). Low occurrence of chromosomal aberration was expected due to the reduction of

cells at metaphase, anaphase and telophase wherein most anomalies occur. Similarly, it could be

also noticed that there was a higher percentage of cells with cytological aberrations (17.14%)

than chromosomal abnormalities (6.99%). Most frequently observed cytological anomalies occur

in the nucleus primarily, vacuolated nucleus (6.33%) and micronucleus (3.93%).

The percentage of aberrations

induced by MT leaf extracts are presented

in Figure 9. The occurrence of

chromosomal aberrations in decreasing

order were stickiness (5.8%), bridges

(0.86%) and fragmentation (0.03%).

Figure 9. The Allium cepa root tip cells
Cytological anomalies were also detected aberrations in different concentrations of Pachira
aquatica leaf extract
mostly in meristematic cells with giant Legend: Orange bars, chromosomal aberrations; Blue bars,
cytological aberrations
(5.8%), strap (4.47%) and vacuolated (3.87%) nuclei. Results also show that there was a greater

percentage of cells with cytoplasmic and nuclear damages (23.71%) than chromosomal

aberrations (6.69%).

According to a study of Pennel

and Lamb (1997), morphological

irregularities observed in cytoplasm

(condensation and shrinkage) and

nucleus (condensation and

margination) were markers that onion

meristematic cells are undergoing
Figure 10. The mitotic cell aberrations triggered
by Pachira aquatica stem and leaf extracts
Mitotic cells at 40x showing aberrant cells (indicated by arrows)
observed in Allium cepa root tips exposed to Pachira aquatica
stem and leaf extracts: A− strap nucleus; B− giant cell,
disintegrated cytoplasm; C− chromosomal bridge; D− nuclear
lesions; E− sticky metaphase; F− giant nucleus; G− ghost cell,
sticky anaphase; H− nuclear erosion
 20
apoptosis. The gathered results suggest that the Allium cepa cells treated with MT extracts that

exhibited cytoplasmic and nuclear abnormalities activated its apoptotic feature to prevent itself

from genetic disruptions.

Chromosome aberrations (See Figure 10) such as stickiness was caused by the

depolymerization of DNA (Darlington, 1942) that suggests the presence of a highly toxic

substance that disrupts chromatin (Fiskesjo, 1985), fragmentation was due to the breakage of its

fragile parts (Chauhan and Chauhan, 1999) and bridges were due to stickiness of chromosomes

which disables the division of cells (Kabarity et al. 1974) and results to mutations in the physical

structure of chromosomes (El-Ghamery et al. 2000). These observed chromosomal aberrations

suggest that the Pachira aquatica extracts could be genotoxic wherein the observed formation of

micronuclei in the onion root tip cells were already early indicators of the genotoxicity of the

extracts (Shahsavan and Samadi, 2001).

21
On the other hand, the percentage

of chromosomal aberrations occurred in

Allium cepa root tip cells exposed from

the lowest to highest concentration (0.63-

3.17%) of stem extract were statistically

similar to Maleic hydrazide (1.3%) which

Figure 11. The Allium cepa root tip cells
suggests the genotoxic action of the aberrations induced by Pachira aquatica stem
extract
phytoconstituents present in the extract. *Means having asterisk as data labels were not
significantly different (p<0.01)
Figure 11 further revealed that the effects of 100 µg/ml (4.20%), 500 µg/ml (4.93%) and 1000

µg/ml (6.20%) in inducing cytological anomalies were comparable with the positive control

(6.17%). Moreover, the over-all occurrence of abnormalities in all extract levels (4.97-6.83%)
have no significant difference with Maleic hydrazide (7.47%) which further showed the potent

cytotoxic mechanisms of MT extracts even at its lowest concentration.

In terms of the percentage of

chromosomal aberrations in each

treatment, root meristems exposed to 100

(0.93%) and 250 µg/ml (0.4%) were

statistically similar to the effects of Maleic

hydrazide which suggests the genotoxicity

Figure 12. The result of onion root tip cells
induced by the high concentrations of MT aberrations test using MT leaf extract
*Means having asterisk as data labels were not
leaf extract. Figure 12 showed that the significantly different (p<0.01)

effects of 100 (3.27%) and 1000 µg/ml (11.27%) in inducing irregularities in nucleus and

cytoplasm were comparable with the positive control. Moreover, the over-all occurrence of

abnormalities at 100 (4.20%) and 1000 µg/ml (13.67%) have no significant difference with the

Maleic hydrazide. The results also revealed that the 500 µg/ml leaf extract was statistically

higher than the negative control in inducing abnormalities in onion cells.

22
In the investigation of Atoyebi et al. (2015) regarding the genotoxicity of Luffa

cylindrica, Nymphaea lotus and Spondias mombin extracts, results showed the frequency of

cellular aberrations that ranges from 0.8-3.5%. Likewise, the cytological and genotoxic action of

Ocimum gratissimum in its lowest concentrations was recorded at 1.4-3.9% (Borooah, 2011).

Percentage of abnormalities induced by Azadirachta indica, Morinda lucida, Cymbopogan

citratus, Mangifera indica and Carica papaya on meristem cells ranges from 0.0-0.14%

(Akinboro and Bakare, 2007). On the other hand, Adegbite et al. (2009) investigated the effects

of neem leaf extract (0.1-0.5 kg/L) on onion meristem cells wherein it was noted with a mean
 percentage of 16.48% in the incidence of abnormalities. In this study, the frequencies of cell

anomalies were noted at 4.97-6.83% in stem extract and 2.47-13.67% in leaf extract thus

exhibiting prominent cytotoxic mechanisms on Allium cepa cells.

Zebrafish Developmental Toxicity Assay (ZDTA)

Zebrafish (Danio rerio) developmental toxicity assay is a reliable vertebrate model and

highly efficient tool for cancer drug prescreening due to its high correlation with the results of

MTT cell proliferation assay (Yigen et al. 2012) since embryogenesis and carcinogenesis have

similar process of progression and most known anticancer drugs are teratogens and vice versa

(Blagosklonny, 2005). Medicinal plants with notable anticancer properties such as Moringa

oleifera, (Goyal et al. 2007), Mangifera indica, Carica papaya, (Chidozie and Adoga, 2014)

were also recorded with teratogenic effects.

A. Embryotoxic Activity
23
Zebrafish embryo lethality was

indicated by coagulation (milky white

appearance), lack of heartbeat, tail

detachment fromyolk sac and absence of

somites (Hood, 2011). Based on the Figure 13. The mortality rate of zebrafish
embryos within different number of hours of
results, the mortality rate of the stem exposure to MT stem extract
*Means having asterisks as data labels have significant
extract was dependent on dose and time of difference with negative control (p<0.01)
hpta – hour post treatment application
exposure. (See Figure 13). It can be noted that as the concentration of MT stem extract increases,

the percentage mortality of Danio rerio embryo also increases since bioactive compounds are

often toxic at higher concentrations (Moshafi et al. 2008).

 In addition, the stem extract was also remarkable with 100% mortality rate after 12 hours

post treatment application (hpta) at 20, 100 and 200 µg/ml concentrations wherein most proven

anticancer drugs induced zebrafish embryo death within 24 hours (Yigen et al. 2012). High

cytotoxicity leading to death was also noted in 2 and 10 µg/ml concentrations at 48 and 24 hpta

respectively.

On the other hand, the Pachira

aquatica leaf extract was also noted with

100% mortality rate in 20, 100 and 200

µg/ml concentrations at 12 hpta. (See

Figure 14). Similarly, the 10 µg/ml leaf

extract led to the death of all embryos after
36 hours. The findings imply the dosage
and time dependence of the lethal effects
Figure 14. The mortality rate of Danio rerio
embryos after exposure to Pachira aquatica leaf
extract in different number of hours
*Means having asterisks as data labels have significant
difference with negative control (p<0.01)
hpta – hour post treatment application

of leaf extract wherein the treatments with 10, 20, 100 and 200 µg/ml of the extract obtained

percentage mortality which were significantly higher (p>0.01) than the control.
24
Different investigations using Carica papaya, (De Castro et al. 2015), Eclipta prostate

and Spilanthes acmella (Ponpornpisit et al. 2011) were documented with 100% mortality of

Danio rerio embryos in its highest concentration after 48 hours of treatment application wherein

its results also showed the embryotoxic action of the extracts used in a dose dependent manner.

Buenaflor et al. (2011) studied the toxicity of A. scholaris wherein its highest concentration was

recorded with lethality after 53 hours of extract exposure. In this study, early embryonic death

was noted at the highest concentration after 12 hours of treatment application even in the lowest

concentration (2 µg/ml) of stem extract which implies the remarkable embryotoxic action of the
phytocompounds. Moreover, Sulik et al. (1988) suggested that this 100% mortality rate among

zebrafish embryos was due to the activation of cell’s apoptotic feature.

According to Ali et al. (2011), the chorion serves as a protective membrane of the

embryo for 48 hours which usually obstruct the penetration of substances particularly drug

permeability. Moreover, compounds with massive molecular weight could not enter the chorion

and limits its embryotoxic activity (JRC Science and Policy Reports, 2014) thus, the high

percentage of zebrafish embryo mortality demonstrates its high bioavailability to possibly

interfere within the structures and mechanisms of zebrafish embryo (Fattorusso and Taglialatela-

Scafati, 2007).

On the other hand, over-all growth

retardation was evidently seen in all

zebrafish embryos treated with 2 and 10

µg/ml of both extract after 24 hours which

further suggest the embryotoxicity and

teratogenicity of the extracts. Figure 15

Figure 15. Growth retardation of zebrafish
revealed at 36 hpta that the embryo in the embryos after 24 hours of extract exposure
*Means having asterisks as data labels have significant
negative control was in the late pharyngula difference with negative control (p<0.01)

stage (Prim-25) while the

25
embryo with 2 µg/ml leaf extract was still in the early

pharyngula stage (Prim-5) which was approximately

10-15 hours’ delay in its development (Kimmel et al.

A B
1995). Coagulation was evident in embryos treated

with 10 and 2 µg/ml leaf extracts at early segmentation

C D
Figure 16. Embryonic development
of Danio rerio with embryo water (A)
and leaf extract (B-D) in 2, 10 and 20
concentrations respectively after 36 h
(1-somite to 3-somite) stage (Kimmel et al. 1995) which indicates the lethal effects of MT

extracts. (See Figure 16).

26
Normal hatching period of zebrafish embryo occurs after 48 hours wherein the formation

of primary organs was in the process of completion (Kimmel et al. 1995) since it is a biomarker

of successful fish embryonic development (De Castro et al. 2015). Figure 17 showed the

hatching success of the treatments wherein 2 µg/ml leaf extract resulted 41.67% hatchability that

was significantly lower (p>0.01) compared to the control. According to Landa and Soldan (n. d.),

effects in hatching varies from partial

suppression of hatching, delay in hatching

and total suppression of hatching. A 12-

hour hatching delay in zebrafish embryos

treated with 2 µg/ml leaf extract was

recorded compared to 100% hatching

Figure 17. The effect of MT stem and leaf extracts
success of embryos at negative control at on the hatchability rate of zebrafish
*Mean having an asterisk as data label has significant
48 hpta wherein this significant difference with negative control (p<0.01)

interference with the hatching process correlates with the incidence of morphological

abnormalities (Orrego et al. 2011). In addition, there were no recorded hatched embryos in all of

the concentrations of stem extract and at 10 µg/ml and higher concentrations of leaf extract

which implies the total suppression on the hatching process of Danio rerio embryos by MT

phytocompounds.

Results show that the induced delay by MT extracts in hatching period affected the

hatching rate success and survival of embryos. Hatching delay often results to weakened embryo

that affects the hatching process wherein Figure 8 further shows the abnormal hatching position
 resulted from the inability of the embryo tail to break out from the chorion (Halden, 2010) and

lower quality of survived embryos with occurrences of malformations (Richards et al. 2000) due

to the potential action of money tree bioactive constituents with enzymatic molecular

mechanisms of the zebrafish (De Castro et al. 2015).

A. Teratogenic Activity
27
37
High frequency of embryonic death previously discussed serves as an early marker of

teratological action (Kusumi and Dunwoodie, 2009). Teratogenic indicators include growth

retardation and malformations in tail, head, heart, notochord, yolk sac and spine (Hood, 2011).

Morphological abnormalities caused Pachira aquatica 2 µg/ml leaf extract at 60 hpta includes

bent tail, bent spine, yolk sac edema, pericardial edema and ocular edema. Embryos exposed to 2

µg/ml stem extract after 36 and 48 hours showed coagulation, unformed tail, unformed head,

detached tail, yolk sac edema and yolk sac not depleted. (See Figure 18). These malformations

observed in Danio rerio embryos were validation of the activation of the cell’s suicide

mechanism (Sulik et al. 1988) that was evidently seen in the high frequency of early embryonic

death in the earlier phase of the experiment.

Moreover, the

teratogenic endpoints induced

by leaf extract were found to be

similar with the embryo

malformations caused by

retinoic acid (bent spine,

pericardial edema and

Figure 18. Phenotypic changes of zebrafish embryos treated
malformed tail), coumarin with money tree stem (below) and leaf (above) extracts at 36-
60 hpta.
Abbreviations: Bs, bent spine (scoliosis); Bt, bent tail; Co, coagulation;
Oe, ocular edema; P, pigmentation; Pe, pericardial edema; Ufh,
unformed head; Ufh, unformed head; Ynd, yolk sac not depleted; and
Yse, yolk sac edema
 (malformation of head, tail and growth retardation), valproic acid (pericardial edema and skeletal

deformities) and warfarin (malformation of tail) which were very potent teratogens and potential

anticancer agents (Wang et al. 2014; Weigt et al. 2012; Aluru et al. 2013).

Teratogenesis molecular mechanisms that possibly occurred due to the cellular

interaction of MT extracts includes disruptions and alteration in DNA sequence and synthesis,

chromosomal abnormalities, mitotic disturbance, enzyme inhibition (Wilson et al. 1973) and

activation or deactivation of normal cellular processes in an unregulated manner (Kusumi and

Dunwoodie, 2009) which often results to reduced cell proliferation, cell death and malformation

in physical features (Wilson et al. 1973) that was evidently seen in the coagulated and deformed

zebrafish embryos treated with MT extracts.

CONCLUSIONS AND RECOMMENDATIONS

Conclusion

With the gathered results in BSLA, ORTCAA, and ZDTA, money tree (Pachira

aquatica) extracts revealed its significant cytotoxic mechanisms through inducing cytostatic and

cytocidal effects which strongly suggests that the bioactive compounds present in the extracts

could exhibit different modes of action in targeting cells. Through appropriate optimization,

dosage and delivery, MT extracts could be a potential source of a plant-derived cytotoxic

substance as a chemotherapeutic drug.

The following conclusions were drawn from the results of the investigation:

1. Percentage mortality of brine shrimp nauplii triggered by the leaf extract (50-80%) was higher

than stem extract (41-53%). However, due to the increasing trend of inhibitory activity of stem

extract to Artemia salina, its estimated median lethal concentration of 104.75 µg/ml was lower
 than LC50 value of leaf extract at 121.69 µg/ml. Still, both extracts were considered toxic

(bioactive) since LC50 values were less than 1000.

29
2. The mitotic index of onion root tip meristem cells exposed to stem extracts in increasing

concentrations were not statistically different while the percentage of dividing cells treated with

1000 µg/ml leaf extract were significantly lower than 100 and 250 µg/ml. Telophase

accumulation was noted in cells at 100 and 250 µg/ml. The two higher concentrations of stem

extract and all leaf extract treated cells were observed at the prophase stage of mitosis. There was

also a general decrease in cells at metaphase and anaphase in both extracts. In addition, there is

no significant difference in the occurrence of chromosomal aberrations in cells with stem extract

at all extract levels including the genotoxic action at 100 and 250 µg/ml leaf extract. Cytological

anomalies in onion cells triggered by 100 and 1000 µg/ml of both extracts were not statistically

different. Overall occurrences of abnormalities at all concentrations of stem extract including the

100 and 1000 µg/ml of leaf extract were not significantly different. Most frequently observed

chromosomal abnormality was stickiness while prominent cytological anomaly was

vacuolization of nucleus.

3. Zebrafish embryo exposed to both extracts after 12 hours at 20, 100, 200 µg/ml caused early

embryonic death wherein 100% mortality was recorded in embryos with stem extract.

Zebrafishes that survived at 0.1% leaf extract were noted with delayed hatching (60 hpta) and

growth retardation. Teratogenic endpoints of leaf extract were bent spine (scoliosis), bent tail and

pericardial edema.

4. The antimitotic activity of stem extract in all concentrations including the 1000 µg/ml leaf extract

were comparable with Maleic hydrazide (positive control) and statistically higher than the

negative control. There is also no significant difference in the genotoxic activity at the highest
 and lowest concentrations of both extracts and positive control. Embryotoxicity and

teratogenicity of the MT leaf and stem extracts were significantly higher than the negative

control.

30
Recommendations

Based on the study conducted, the following recommendations are hereby drawn:

1. Utilization of bark, flowers, seeds, roots and fruits of money tree as potential sources of

cytotoxic compounds.

2. Isolation, purification and identification of specific bioactive compounds that are

responsible for the cytotoxic activity of Pachira aquatica stem and leaf extract.

3. Variation of extraction procedures to detect the presence of all phytochemical

constituents of Pachira aquatica.

4. Further investigation and validation of the effect of the extracts using human cancer cells

and other mammalian test systems.

5. Investigation of other biological activities of Pachira aquatica stem and leaf extracts
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ACKNOWLEDGEMENT

First and foremost, we would like to thank God Almighty for giving us the chance to

make this study possible and for the wisdom and strength that He gave throughout this research.

This piece of work is wholeheartedly dedicated for the glory of His Name.

To Dr. Estrelita S. Cunanan, Schools Division Superintendent of Gapan City, Dr. Paulina

C. Perez, Education Program Specialist in Science and Mrs. Elvira A. Pascua, Principal IV of

JRLMHS for authorizing us to undertake this pursuit of knowledge.

 Deepest thanks to Ms. Nenita C. Manalastas, Science Department Head Teacher for her

unending efforts, encouragement and support. We would also like to thank our research teacher,

Dr. Wajavina N. Catacutan, for sharing her immense knowledge about research.

We would like to express our gratitude and love to our parents, for their financial support

and unceasing encouragements. They gave us strength to chase for our dreams and unceasingly

strive on whenever difficulties arise.

To our classmates and friends, for reminding us to smile whenever we are facing

difficulties in the experimentation. And to everyone who provide valuable contributions in the

accomplishment of this study, deepest thanks to all of you! Dios mamajes!

Jane Nicole N. Catacutan

Melissa C. Gaudario
Maries Ann R. Silvestre

41

CURRICULUM VITAE

Name: Jane Nicole Nagaño Catacutan

Address: Nieves, San Leonardo, Nueva Ecija
Birthday: November 23, 1999
Birthplace: Gapan City
Name of Father: Nelson M. Catacutan
Name of Mother: Wajavina N. Catacutan
Name of Brother/Sister: Janel Rose N. Catacutan
: Nielsen N. Catacutan

Educational Background
a. Pre-elementary
Good Shepherd’s Foundation Learning Center
Nieves, San Leonardo
 S.Y. 2004- 2006
b. Elementary
Mambangnan Elementary School
Mambangnan, San Leonardo
S.Y. 2006-2012
c. High School
Juan R. Liwag Memorial High School
Bayanihan, Gapan City
S.Y. 2012-present

Favorite Subject: Science and Technology

Ambition: To be a Medical Doctor
Philosophy in life: Be not afraid of going slowly. Be afraid of standing still.

42
CURRICULUM VITAE

Name: Melissa Candelaria Gaudario

Address: San Vicente, Gapan City, Nueva Ecija
Birthday: July 29, 1999
Birthplace: San Vicente. Gapan City, Nueva Ecija
Name of Father: Petronilo P. Gaudario
Name of Mother: Norayda C. Gaudario
Name of Brother/Sisters: Darwin C. Gaudario
: Kristine C. Gaudario
: Ramon C. Gaudario
: Paulo C. Gaudario
: Lorie Laine C. Gaudario
: Ronilo C. Gaudario

Educational Background
a. Pre-elementary
Gapan North Central School
San Vicente, Gapan City
S.Y. 2004- 2006
b. Elementary
Gapan North Central School
San Vicente, Gapan City
S.Y. 2006-2012
c. High School
Juan R. Liwag Memorial High School
Bayanihan, Gapan City
S.Y. 2012-present

Favorite Subject: Science

Ambition: To be a medical doctor

Philosophy in Life: “Everything is going to be alright maybe not today but eventually, just have
faith and be thankful because where faith and hope grows, miracle blossoms”

43
CURRICULUM VITAE

Name: Maries Ann Ramos Silvestre

Address: Riverview Drive, San Nicolas, Gapan City
Birthday: August 1, 2000
Birthplace: Gonzales General Hospital
Name of Father: Moises B. Silvestre Jr.
Name of Mother: Marian R. Silvestre
Name of Brother: Moises Marion R. Silvestre

Educational Background
a. Pre-elementary:
General De Jesus College
Poblacion, San Isidro, Nueva Ecija
S.Y. 2003-2006
b. Elementary:
General De Jesus College
Poblacion, San Isidro, Nueva Ecija
S.Y. 2007-2013

c. High School:
Juan R. Liwag Memorial High School
Bayanihan, Gapan City
S.Y. 2013-present

Favorite Subject: Mathematics and Biotechnology

Ambition: To be a Dentist
Philosophy in Life: “Be the change you wish to see in this world”

APPENDICES
Image 1.1 Weighing of 38 grams of Sodium Chloride

Image 1.2 Preparation of Artificial Sea Water

45
Image 1.3 Spawning of Brine Shrimps eggs for 48 hours

Image 1.4 Dissolving of extracts in DMSO

46
Image 1.5 Preparation of dissolved extracts into different concentrations

Image 1.6 Different concentrations of leaf and stem extracts

47
Image 1.7 Transferring of the different concentrations of extracts into the well plates

Image 1.8 Transferring of the Brine Shrimp nauplii using a micropipette into the 24-well
plates 48
Image 1.9 Brine Shrimps nauplii exposed with different treatments for 24 hours.

49
 Image 2.1 Weighing 100 µg of extracts using analytical balance

Image 2.2 Dissolving 100 µg of extracts in 1 ml of Dimethyl sulfoxide (DMSO)

50
 Image 2.3 Preparation of Onions

Image 2.4 Preparation of Treatments

51
Image 2.5 Transferring of the prepared treatments into the bottles

Image 2.6 Exposure of onions in different treatments 52

Image 2.7 Preparation of Farmer’s Fluid with 1:3glacial acetic acid and ethanol ratio

Image 2.8 Transferring of Farmer’s Fluid in micro tubes

 53

Image 2.9 Cutting of Onion Root Tips and placing it into the micro tubes with Farmer’s Fluid for
fixation and storage in refrigerator for 24 hours

Image 2.10 Placing of Onion Root Tips in glass slides

 54

Image 2.11 Dropping of 1N HCL (Hydrochloric Acid) into the Onion Root Tip

Image 2.12 Maceration of Onion Root Tips using a scalpel

 55

Image 2.13 Placing One Drop of Aceto-Orcein on the Crushed Root tip

Image 2.14 Heating the Slide Gently Over the Flame of the Alcohol Lamp
 56

Image 2.15 Covering the Stained Root Tip with a Cover Slip

Image 2.16 Pressing the Cover Slip Firmly to squash onion cells and remove excess stain
 57

Image 2.17 Preparation of Slides

Image 2.18 Microscopy of Slides

 58

Image 3.1 Preparation of the Aquarium

Image 3.2 Localization of zebrafish in a plastic mesh inside the Aquarium

 59

Image 3.3 Covering the Aquarium with a Black Plastic Bag to induce spawning

Image 3.4 Eggs were exposed to lighted conditions to trigger spawning

 60

Image 3.5 The eggs were siphoned out using a hose and aspirator bulb

Image 3.6 Transferring the Eggs in a Petri Dish

 61

Image 3.7 Exposing of Zebrafish in different treatments

Image 3.8 Gathering of Eggs for microscopy from well plates using a droppe
 62

Image 4. Plant Authentication of Money Tree (Pachira aquatica)

Image 5 Phytochemical Analysis of Money Tree Stem Extract
63
 64

Image 6. Phytochemical Analysis of Money Tree Leaves Extract

65
 66

Image 7. Extract Yield of the Stem of Money Tree

 67

Image 8 Extract Yield of the Leaves of Money Tree

 68

3.125 µg/ml 6.25 µg/ml 12.5 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml
R1 44.77 R1 30.96 R1 44.77 R1 74.89 R1 100 R1 44.77
R2 17.15 R2 15.03 R2 24.68 R2 44.87 R2 17.15 R2 38.63
R3 17.15 R3 -0.42 R3 17.15 R3 17.15 R3 38.63 R3 7.94
R4 38.63 R4 7.94 R4 44.17 R4 72.38 R4 44.77 R4 -10.47
R5 72.38 R5 44.77 R5 -38.08 R5 44.77 R5 72.38 R5 44.77
R6 -38.08 R6 -38.08 R6 74.89 R6 100 R6 30.96 R6 74.89
R7 53.97 R7 100 R7 24.68 R7 82.74 R7 -10.47 R7 100
R8 53.97 R8 53.97 R8 72.38 R8 76.99 R8 49.79 R8 100
R9 53.97 R9 100 R9 44.77 R9 24.68 R9 57.51 R9 76.99
R10 100 R10 57.51 R10 100 R10 -0.42 R10 100 R10 -10.47
R11 -0.42 R11 72.38 R11 36.27 R11 76.99 R11 44.77 R11 38.63
R12 100 R12 49.79 R12 69.31 R12 30.96 R12 17.15 R12 72.38
Mean 42.79 Mean 41.15 Mean 42.97 Mean 53.82 Mean 46.89 Mean 48.17

Table 2 Percentage Mortality of Brine Shrimp Nauplii treated with MT Stem Extract

3.125 µg/ml 6.25 µg/ml 12.5 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml
R1 17.92 R1 17.92 R1 100 R1 100 R1 75.13 R1 100
R2 72.64 R2 100 R2 100 R2 45.28 R2 45.28 R2 45.28
R3 69.6 R3 21.83 R3 17.92 R3 100 R3 72.64 R3 21.28
R4 45.28 R4 100 R4 100 R4 100 R4 72.64 R4 100
R5 60.91 R5 72.64 R5 100 R5 100 R5 75.13 R5 72.64
R6 69.6 R6 72.64 R6 100 R6 45.28 R6 100 R6 17.92
R7 77.2 R7 77.2 R7 77.2 R7 54.4 R7 75.13 R7 31.6
R8 36.86 R8 57.91 R8 75.13 R8 100 R8 72.64 R8 72.64
R9 45.28 R9 72.64 R9 100 R9 100 R9 8.8 R9 8.8
R10 50.25 R10 50.25 R10 75.13 R10 50.25 R10 54.4 R10 8.8
R11 50.25 R11 57.91 R11 31.6 R11 75.13 R11 57.91 R11 72.64
R12 57.91 R12 72.64 R12 54.4 R12 100 R12 25.38 R12 45.28
Mean 54.48 Mean 64.47 Mean 77.61 Mean 80.86 Mean 61.26 Mean 49.79

Table 3 Percentage Mortality of Brine Shrimp Nauplii treated with MT Leaf Extract
 69
MITOTIC INDICES MITOTIC PHASE INDICES
P M A T TOTAL MI (%) P M A T TOTAL MI (%)
0 240 16 96 304 656 21.87 0 36.59 2.44 14.63 46.34 656 21.87
100 44 0 4 176 224 7.47 100 19.64 0.00 1.79 78.57 224 7.47
250 48 8 16 168 240 8.00 250 20.00 3.33 6.67 70.00 240 8.00
500 214 6 6 18 244 8.13 500 87.70 2.46 2.46 7.38 244 8.13
1000 244 8 8 12 272 9.07 1000 89.71 2.94 2.94 4.41 272 9.07
Positive 150 10 10 16 186 6.20 Positive 80.65 5.38 5.38 8.60 186 6.20

PROPHASE R1 R2 R3 Total ANAPHASE R1 R2 R3 Total TOTAL R1 R2 R3 Total

0 82 84 74 240 0 23 34 39 96 0 211 235 210 656
100 10 18 16 44 100 2 1 1 4 100 63 83 78 224
250 14 22 12 48 250 4 8 4 16 250 74 69 97 240
500 74 62 78 214 500 0 6 0 6 500 88 75 81 244
1000 82 72 90 244 1000 2 2 4 8 1000 86 82 104 272
Positive 42 48 60 150 Positive 3 1 6 10 Positive 53 54 79 186

METAPHASE R1 R2 R3 Total TELOPHASE R1 R2 R3 Total TOTAL R1 R2 R3 Mean (%)

0 4 5 7 16 0 102 112 90 304 0 21.10 23.50 21.00 21.87
100 0 0 0 0 100 51 64 61 176 100 6.30 8.30 7.80 7.47
250 2 0 6 8 250 54 39 75 168 250 7.40 6.90 9.70 8.00
500 2 4 0 6 500 12 3 3 18 500 8.80 7.50 8.10 8.13
1000 2 4 2 8 1000 0 4 8 12 1000 8.60 8.20 10.40 9.07
Positive 3 3 4 10 Positive 5 2 9 16 Positive 5.30 5.40 7.90 6.20

Table 4 Raw data of Counted Onion Cells treated with MT Stem Extract

MITOTIC INDICES MITOTIC PHASE INDICES

P M A T TOTAL MI (%) P M A T TOTAL MI (%)
0 240 16 96 304 656 21.87 0 36.59 2.44 14.63 46.34 656 21.87
100 360 48 48 48 504 16.80 100 71.43 9.52 9.52 9.52 504 16.80
250 300 54 60 48 462 15.40 250 64.94 11.69 12.99 10.39 462 15.40
500 188 22 22 70 302 10.07 500 62.25 7.28 7.28 23.18 302 10.07
1000 120 40 60 34 254 8.47 1000 47.24 15.75 23.62 13.39 254 8.47
Positive 150 10 10 16 186 6.20 Positive 80.65 5.38 5.38 8.60 186 6.20

PROPHASE R1 R2 R3 Total ANAPHASE R1 R2 R3 Total TOTAL R1 R2 R3 Total

0 82 84 74 240 0 23 34 39 96 0 211 235 210 656
100 121 134 105 360 100 21 12 15 48 100 177 174 153 504
250 120 109 71 300 250 19 17 24 60 250 166 164 132 462
500 56 63 69 188 500 8 6 8 22 500 104 89 109 302
1000 34 47 39 120 1000 24 21 15 60 1000 80 91 83 254
Positive 42 48 60 150 Positive 3 1 6 10 Positive 53 54 79 186

METAPHASE R1 R2 R3 Total TELOPHASE R1 R2 R3 Total TOTAL R1 R2 R3 Mean (%)

0 4 5 7 16 0 102 112 90 304 0 21.10 23.50 21.00 21.87
100 13 15 20 48 100 22 13 13 48 100 17.70 17.40 15.30 16.80
250 16 21 17 54 250 11 17 20 48 250 16.60 16.40 13.20 15.40
500 9 8 5 22 500 31 12 27 70 500 10.40 8.90 10.90 10.07
1000 13 11 16 40 1000 9 12 13 34 1000 8.00 9.10 8.30 8.47
Positive 3 3 4 10 Positive 5 2 9 16 Positive 5.30 5.40 7.90 6.20

Table 5 Raw data of Counted Onion Cells treated with MT Leaf Extract
 70
ABERRANT CELLS
ABERRATIONS 1000 500 250 100 Total
Fragmented
2 0 0 0 2
chromosomes
Chromosome
5 16 57 48 126
Stick iness
Chromosome
12 4 38 28 82
bridges
Vacuolated
54 70 2 64 190
nucleus
Binucleated cell 6 2 2 10 20
Strap nucleus 24 10 10 8 52
Nuclear erosion 28 20 8 0 56
Giant cell 0 0 4 20 24
Micronucleus 50 40 22 6 118
Disintegrated
0 0 0 2 2
cytoplasm
Giant nucleus 20 6 6 16 48
Ghost cells 4 0 0 0 4
TOTAL 205 168 149 202 724

Table 6 Raw data of Counted Aberrant Cells induced by MT Stem Extract

Chromosomal Aberrations (LEAF)

ABERRATIONS 1000 500 250 100 Total
Fragmented
0 0 0 1 0
chromosomes
Chromosome
82 58 10 24 174
stick iness
Chromosome
6 14 2 4 26
bridges
Vacuolated nucleus 0 70 36 10 116

Binucleated cell 18 24 8 20 70
Strap nucleus 72 42 4 16 134
Nuclear erosion 8 34 2 8 52
Giant cell 16 32 0 0 48
Micronucleus 0 28 6 44 78
Disintegrated
0 50 0 0 50
cytoplasm
Giant nucleus 100 58 6 0 164
Ghost cells 0 0 0 1 0
TOTAL 302 410 74 128 914

Table 7 Raw data of Counted Aberrant Cells induced by MT Leaf Extract

 71

Anova: Single Factor a 0.01

LSD 3.102248
SUMMARY HSD 4.381439
Groups Count Sum Average Variance Scheffe 3.232267
0 3 65.60 21.87 2.003333 Post Hoc 0 100 250 500 1000
100 3 22.40 7.47 1.083333 100 14.4
250 3 24.00 8.00 2.23 250 13.86667 0.533333
500 3 24.40 8.13 0.423333 500 13.73333 0.666667 0.133333
1000 3 27.20 9.07 1.373333 1000 12.8 1.6 1.066667 0.933333
Positive 3 18.60 6.20 2.17 Positive 15.66667 1.266667 1.8 1.933333 2.866667
Colored cells have signficant mean differences

ANOVA Reject Null Hypothesis because p < 0.01 (Means are Different)
Source of Variation SS df MS F P-Value F crit
Between Groups 509.8244 5 101.9649 65.9019 0.000 5.064343
Within Groups 18.56667 12 1.547222

Total 528.3911 17

Table 8 ANOVA Table of Mitotic indices of Onion Cells treated with

MT Stem Extract

Anova: Single Factor a 0.01

LSD 3.353302
SUMMARY HSD 4.736013
Groups Count Sum Average Variance Scheffe 3.493844
0 3 65.60 21.87 2.003333 Post Hoc 0 100 250 500 1000
100 3 50.40 16.80 1.71 100 5.066667
250 3 46.20 15.40 3.64 250 6.466667 1.4
500 3 30.21 10.07 1 500 11.79667 6.73 5.33
1000 3 25.40 8.47 0.323333 1000 13.4 8.333333 6.933333 1.603333
Positive 3 18.60 6.20 2.17 Positive 15.66667 10.6 9.2 3.87 2.266667
Colored cells have signficant mean differences

ANOVA Reject Null Hypothesis because p < 0.01 (Means are Different)
Source of Variation SS df MS F P-Value F crit
Between Groups 522.2587 5 104.4517 57.77908 0.000 5.064343
Within Groups 21.69333 12 1.807778

Total 543.952 17

Table 9 ANOVA Table of Mitotic indices of Onion Cells treated with

MT Leaf Extract
 72

Anova: Single Factor a 0.01

LSD 2.543118
SUMMARY HSD 3.481588
Groups Count Sum Average Variance Scheffe 2.778386
100 3 7.59 2.53 1.1317 Post Hoc 100 250 500 1000
250 3 9.5 3.166667 3.210833 250 0.636667
500 3 2 0.666667 0.413333 500 1.863333 2.5
1000 3 1.9 0.633333 0.063333 1000 1.896667 2.533333 0.033333
Positive 3 3.9 1.3 0.01 Positive 1.23 1.866667 0.633333 0.666667
Colored cells have signficant mean differences

ANOVA Cannot Reject Null Hypothesis because p > 0.01 (Means are the same)
Source of Variation SS df MS F P-Value F crit
Between Groups 15.59189 4 3.897973 4.035838 0.033 5.994339
Within Groups 9.6584 10 0.96584

Total 25.25029 14

Table 10 ANOVA Table of Chromosomal Aberrations of Onion Cells treated

with MT Stem Extract

Anova: Single Factor a 0.01

LSD 3.999321
SUMMARY HSD 5.475163
Groups Count Sum Average Variance Scheffe 4.369305
100 3 12.6 4.2 4.89 Post Hoc 100 250 500 1000
250 3 5.4 1.8 0.13 250 2.4
500 3 14.8 4.933333 2.763333 500 0.733333 3.133333
1000 3 18.6 6.2 3.43 1000 2 4.4 1.266667
Positive 3 18.51 6.17 0.7297 Positive 1.97 4.37 1.236667 0.03
Colored cells have signficant mean differences

ANOVA Cannot Reject Null Hypothesis because p > 0.01 (Means are the same)
Source of Variation SS df MS F P-Value F crit
Between Groups 39.35283 4 9.838207 4.118806 0.032 5.994339
Within Groups 23.88607 10 2.388607

Total 63.23889 14

Table 11 ANOVA Table of Cytological Aberrations of Onion Cells treated

with MT Stem Extract
 73

Anova: Single Factor a 0.01

LSD 4.287479
SUMMARY HSD 5.869658
Groups Count Sum Average Variance Scheffe 4.68412
100 3 20.19 6.73 4.4607 Post Hoc 100 250 500 1000
250 3 14.9 4.966667 2.743333 250 1.763333
500 3 16.8 5.6 4.44 500 1.13 0.633333
1000 3 20.5 6.833333 1.703333 1000 0.103333 1.866667 1.233333
Positive 3 22.41 7.47 0.3787 Positive 0.74 2.503333 1.87 0.636667
Colored cells have signficant mean differences

ANOVA Cannot Reject Null Hypothesis because p > 0.01 (Means are the same)
Source of Variation SS df MS F P-Value F crit
Between Groups 12.31207 4 3.078017 1.12123 0.400 5.994339
Within Groups 27.45213 10 2.745213

Total 39.7642 14

Table 12 ANOVA Table of Overall Aberrant Onion Cells treated with

MT Stem Extract

Anova: Single Factor a 0.01

LSD 0.978137
SUMMARY HSD 1.339092
Groups Count Sum Average Variance Scheffe 1.068626
100 3 2.79 0.93 0.0637 Post Hoc 100 250 500 1000
250 3 1.2 0.4 0.07 250 0.53
500 3 7.2 2.4 0.13 500 1.47 2
1000 3 8.79 2.93 0.4107 1000 2 2.53 0.53
Positive 3 3.9 1.3 0.04 Positive 0.37 0.9 1.1 1.63
Colored cells have signficant mean differences

ANOVA Reject Null Hypothesis because p < 0.01 (Means are Different)
Source of Variation SS df MS F P-Value F crit
Between Groups 13.16244 4 3.29061 23.03059 0.000 5.994339
Within Groups 1.4288 10 0.14288

Total 14.59124 14

Table 13 ANOVA Table of Chromosomal Aberrations of Onion Cells treated

with MT Leaf Extract
 74
Anova: Single Factor a 0.01
LSD 3.958336
SUMMARY HSD 5.419053
Groups Count Sum Average Variance Scheffe 4.324527
100 3 9.81 3.27 0.6517 Post Hoc 100 250 500 1000
250 3 6.21 2.07 0.1197 250 1.2
500 3 33.81 11.27 5.6017 500 8 9.2
1000 3 21.39 7.13 5.1517 1000 3.86 5.06 4.14
Positive 3 18.51 6.17 0.1747 Positive 2.9 4.1 5.1 0.96
Colored cells have signficant mean differences

ANOVA Reject Null Hypothesis because p < 0.01 (Means are Different)
Source of Variation SS df MS F P-Value F crit
Between Groups 155.9246 4 38.98116 16.65933 0.000 5.994339
Within Groups 23.399 10 2.3399

Total 179.3236 14

Table 14 ANOVA Table of Cytological Aberrations of Onion Cells treated

with MT Leaf Extract

Anova: Single Factor a 0.01

LSD 4.967293
SUMMARY HSD 6.800338
Groups Count Sum Average Variance Scheffe 5.426825
100 3 12.6 4.2 0.57 Post Hoc 100 250 500 1000
250 3 7.41 2.47 0.1267 250 1.73
500 3 41.01 13.67 16.1077 500 9.47 11.2
1000 3 30.18 10.06 0.1468 1000 5.86 7.59 3.61
Positive 3 22.41 7.47 1.4727 Positive 3.27 5 6.2 2.59
Colored cells have signficant mean differences

ANOVA Reject Null Hypothesis because p < 0.01 (Means are Different)
Source of Variation SS df MS F P-Value F crit
Between Groups 242.3608 4 60.59019 16.44337 0.000 5.994339
Within Groups 36.8478 10 3.68478

Total 279.2086 14

Table 15 ANOVA Table of Overall Aberrant Onion Cells treated with

MT Leaf Extract
 75
Mortality (%) Hatchability (%)
12H 60H
Conc. R1 R2 R3 Mean Conc. R1 R2 R3 Mean
Negative 0 0 0 0 Negative 100 100 100 100
2.0 25 0 0 8.33 2.0 0 0 0 0
10.0 100 100 50 83.33
20.0 100 100 100 100
100.0 100 100 100 100
200.0 100 100 100 100
Growth retardation (%)
24H 24H
Conc. R1 R2 R3 Mean Conc. R1 R2 R3 Mean
Negative 0 0 0 0 Negative 0 0 0 0
2.0 25 0 25 16.67 2.0 100 100 100 100
10.0 100 100 100 100

36H 48H
Conc. R1 R2 R3 Mean Conc. R1 R2 R3 Mean
Negative 0 0 0 0 0.0 0 0 0 0
2.0 100 0 75 58.33 0.1 100 100 100 100

Table 16 Raw data of Mortality Rate, Hatchability and Growth Retardation

Mortality (%) Hatchability (%)

12H 60H
Conc. R1 R2 R3 Mean Conc. R1 R2 R3 Mean
Negative 0 0 0 0 Negative 100 100 100 100
2.0 0 0 0 0 2.0 25 50 50 41.67
10.0 50 50 50 50
20.0 100 100 100 100
100.0 100 100 100 100
200.0 100 100 100 100 Growth retardation (%)
24H
24H Conc. R1 R2 R3 Mean
Conc. R1 R2 R3 Mean Negative 0 0 0 0
Negative 0 0 0 0 2.0 50 75 75 66.67
2.0 0 25 0 8.33 10.0 100 100 100 100.00
10.0 100 100 50 83.33

36H
Conc. R1 R2 R3 Mean 48H
Negative 0 0 0 0 Conc. R1 R2 R3 Mean
2.0 0 25 0 8.33 Negative 0 0 0 0
10.0 100 100 100 100.00 2.0 0 25 0 8.33

Table 17 Raw data of Mortality Rate, Hatchability and Growth Retardation