RESEARCH PLAN

Project Title: Comparison of Cytotoxic effects between Mimosa pudica, Leucaena

leucocephala, Albizia saman leaves using brine shrimp assay

Project Proponent/s: John Rey Robles Dilig

John Christian Jerick Giron

Sean Ezekiel De leon Salvador

Project Adviser / Adult Sponsor: Elaine Mariano Guaza

RATIONALE

Synopsis of the Background of Study

1. Cytotoxicity studies are useful initial step to know the potential toxicity of a certain

substances. It can be use in pharmaceutical and cosmetic preparation but this study has a

crucial role (McGaw, Elgorashi & Eloff, 2014).

2. Cell cytotoxicity refers to the ability of a certain chemical or substances to destroy

living cells (Green, 2012).

3. The researchers came up with the idea on comparing plants that has the same family

(Fabaceae) and sub – family (Samanea) and determining the difference in its cytotoxic

effects on brine shrimps using mimosine.

4. The researchers used Mimosa pudica, Lucena leucocephala, and Albizia Saman and

their leaves in determining their cytotoxic properties.

5. The first plant, Mimosa pudica or shameplantis a creeping annual or perennial herb. It

has been found to have antiasthmatic, aphrodisiac, analgesic and antidepressant

(Gandhiraja et al., 2009).

6. Ngo Bum et al., (2018) defined M. pudica as a common plant moist waste ground,

lawns, open plantations and weedy thickets.Akter et al., (2010) proved that M. pudicahas

antibacterial properties against 15 Grams-positive and Gram-negative human pathogenic

bacteria.

7. The second plant, L. leucocephala is known as the 'miracle tree' because of its

worldwide success as a long-lived and highly nutritious forage tree, and its great variety

of other uses.

8. Lucaena luecocephala, despite the presence of inherent anti-nutritional factors such as

tannin and mimosine has invariably limited its utilization by the livestock (Onwuka et al.,

1992; Ajit et al., 2010). Mimosine which is a non-protein amino acid structurally exerts

its toxic action by blocking the metabolic pathways of aromatic amino acids and

tryptophan (D’Mello, 2002).

9. The third plant, Albizia saman (Leguminosae) is rich in alkaloids and globally

distributed throughout the tropical regions. The leaves are used in folk remedy for

stomach cancer, common cold, diarrhoea, headache, intestinal ailments, sore throat,

stomach ache, and wounds (Prasad et al., 2010).

10. Some biological activities of this plant have been reported in aqueous and solvent

extracts (3-5, 10, 17, 19, 24)(Arulpriya et al., 2010, Arumugam et al. 2011, Azhar et al.,

2009), Reza and phytochemical analysis of this species revealed pithecolobine as the

main alkaloid.

Societal Impacts of the Study

1. This study can help in determining the difference or similarities between the plants

given.
 2. This study can help determine which plant has higher concentrations of mimosine.

3. The analysis of cytotoxic effects on M. pudica, L. leucocephala, and A. saman leaves

extract gives advantage to medical fields, because the chemical that the researchers are

going to test which is mimosine is a potential inhibitor to cancer cells.

STATEMENT OF THE OBJECTIVES/PROBLEMS

This study focuses on the following research objectives namely:

1. To determine the mortality rate and survival rate with the various concentrations of

fresh and dried M. pudica, L. leucocephala, and A. saman leaves.

2. To determine which leaves have a higher concentration of mimosine to know which

plant can kill the most number of nauplii.

3. To relate the effects of various concentrations and the extract used to the fresh and

dried M. pudica, L. leucocephala, and A. saman leaves.

HYPOTHESIS OF THE STUDY

This study will work out on the following hypothesis, namely:

Ho

1. There is no significant difference between the fresh and dried M. pudica, L.

leucocephala, and A. saman leaves in terms of mortality rate and survival rate of nauplii.

2. There is no significant difference between the concentrations of mimosine to the

number of dead nauplii.

3. There is no relation in the effects of various concentrations and the extract used to

fresh and dried M. pudica, L. leucocephala, and A. saman leaves.

 Ha

1. There is a significant difference between the fresh and dried M. pudica, L.

leucocephala, and A. saman leaves in terms of mortality rate and survival rate of nauplii.

2. There is a significant difference between the concentrations of mimosine to the number

of dead nauplii.

3. There is a relation in the effects of various concentrations and the extract used to fresh

and dried M. pudica, L. leucocephala, and A. saman leaves.

EXPECTED OUTCOME(S)

This research study will aim to accomplish the following outcomes, namely:

1. A more in-depth study on the relationship of the survival rate and mortality rate of

nauplii.

2. Determination of the different relationships of various concentrations of mimosine to

the number of nauplii.

3. Dried leaves having more concentration of mimosine than fresh leaves.

PROCEDURE

Preparation of Brine Shrimp Lethality Statistical Analysis and

Extracts, Controls and Assay (BSLA) Proper Logit-Probit Regression
Samples Model
of Extracts, Controls

and Samples
Preparation of Extracts, Controls and Samples

A total of one (1) kg of A. saman (Jacq.) Merr. Leaves, M. pudica Linn leaves,

and L. leucocephala (Lam.) de Wit leaves will be gathered from Loma de Gato, Marilao,
Bulacan and Prenza I, Marilao Bulacan. One set of the leaves will be dried. The other set

of leaves will be fresh. The leaves will be cleaned thoroughly by distilled water to

remove dusts and other debris. For preparation of dried foliar extract the set of leaves

such as A. saman, L. leucocephala and M. pudica measuring five hundred grams (500g)

will be air-dried for one (1) at room temperature. After air-drying, the dried leaves will be

ground by the kitchen blender for the preparation of stock solution. For the preparation of

fresh foliar extract, the other set of leaves measuring five hundred grams (500g) will be

blended by the kitchen blender. The ground dried leaves will be soaked in one thousand

millilitres (1000mL) of artificial sea water. The blended fresh leaves will be soaked in

one thousand five hundred millilitres (1500mL) of artificial sea water.

Several treatments of control setups will be prepared such as 2% of

ethanol (positive control), artificial salt water (negative control) and 0.45% dimethyl

sulfoxide (vehicle control). Artificial sea water will be prepared by dissolving twenty

grams (20 g) of rock salt in five hundred forty milliliters (540 mL) distilled water. Two

percent ethanol will be prepared by diluting of two milliliters (2 mL) of 95% ethanol to

one hundred milliliters (100 mL) distilled water. The 0.45% DMSO solution will then be

prepared by diluting four milliliters (4.5 mL) dimethyl sulfoxide to one liter (1 L)

distilled water.

The stock solutions from dried and fresh A. saman, L. leucocephala and M.

pudica leaves will be incubated for twenty four hours (24 h) at room temperature and

filtered after the said incubation period. After incubation, different concentrations (T1 =

100 µg/mL, T2 = 50 µg/mL, T3 = 25 µg/mL, T4 = 12.5 µg/mL, T5 = 6.25 µg/mL, T6 = 0

µg/mL) will be prepared through serial dilution setup using a screw cap centrifuge tubes.
Brine Shrimp Lethality Assay (BSLA) Proper

The Brine Shrimp Lethality Assay (BSLA) developed by Michael and his

colleagues (1956) will be conducted using the larvae (nauplii) of Artemiasalina as the test

organism for determining the lethal dose concentration (LC50) of A. saman, L

leucocephala and M. pudica. According to the assasy proper, one-eight (1/8) spatula of

brine shrimp eggs will be placed on artificial sea water (ASW). In order to hatch the eggs,

the setup will be aerated and illuminated for a span of about twenty-four hours (24h). A

light source will be placed on one side of the setup container to attract the hatched nauplii

after the 24-hour incubation period.

A twelve (12) hole multi-well plate will be used to contain the treatment and

controls for the Brine Shrimp Lethality Assay. Each well of the plate will be filled with

five millilitres (5 mL) of the prepared solutions of varying concentrations from dried and

fresh foliar extract stock solution. Twelve (12) replicate wells will be prepared for each

sample concentration and controls for verification purposes.

Ten (10) nauplii will be transferred carefully in each of the well using a Pasteur

pipette. Each nauplii-containing wells will then be incubated again for twenty four hours

(24 h) at room temperature. After 24-hour incubation period, each wells will be checked

using a magnifying glass and the surviving nauplii will be counted. A simple test will be

conducted to see how many naupliis are alive and dead. In this case, if the nauplii does

not move for about thirty seconds (30 s) then it will be considered dead. In addition to

that, nauplii turn opaque when dead.

 The percentage mortality will be calculated at each concentrations and samples

treatments. This variable for each well will be determined using the formula:

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑛𝑎𝑢𝑝𝑙𝑖𝑖

% 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 = 𝑥 100
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑎𝑢𝑝𝑙𝑖𝑖

Statistical Tools: Two-way Analysis of Variance (ANOVA) and Logit-Probit

Regression Model

The data recorded from the bioassay will be subjected to two-way analysis of

variance (ANOVA) to determine the significant effects and interaction of the independent

variables, namely: type of extracts (dried and fresh A. saman, L. leucocephala and M.

pudica foliar extracts) and treatment concentrations (T1 = 100 µg/mL, T2 = 50 µg/mL, T3

= 25 µg/mL, T4 = 12.5 µg/mL, T5 = 6.25 µg/mL, T6 = 0 µg/mL), to the dependent

variables such as the percentage mortality and the lethal dose concentration (LC50) of the

plant extracts.

The concentration-mortality data will be analyzed using the Logit-Probit

Regression Model. This analysis will be used to indicate the effectiveness and phytotoxic

potency of a certain plant extract by determining the lethal dose concentration (LC50).

The LC50 value represents the concentration of the chemical had produced death in half

of the test subjects, in this case the brine shrimp, after certain period of exposure (Pillai &

Nair, 2013).

DATA ANALYSIS

The data that will be obtained from the Brine Shrimp Lethality Assay of the Albizia

saman (Jacq.) Merr., M. pudica Linn, and L. leucocephala (Lam.) de Wit extracts will be
subjected to descriptive and inferential statistics to determine the significant relationship of the

independent and dependent variables. Two-way Analysis of Variances (ANOVA) will be used to

determine the effects and interaction between the independent variables consisting of treatment

types at varying concentration to the dependent variables consisting of the mortality parameters.

DISCUSSION OF RESULTS

Data that will be gathered from the experiment will be tabulated and analyzed. Bar

graphs will be used as the graphical representation of data to show variables such as the total

number of dead nauplii and mortality rate. The graphs will be used to show the different data sets

obtained from the Brine Shrimp Lethality Assay. Logi-Probit Regression Model will use a line

graph to determine the LC50 for each type of treatment.

These data will also be used to determine the significant effects of the independent

variables consisting of treatment types at varying concentration to the dependent variables such

as the mortality rate from the total number of dead nauplii.

CONCLUSION

The conclusion will be focus on the analysis of the relationship of treatment types

consisting of the dried and fresh foliar extracts obtained from Albizia saman(Jacq.) Merr. Also

the conclusion will determine that the changes in the mortality parameterscan determine the

cytotoxic property of of A. saman (Jacq.) Merr., M. pudica Linn, and L. leucocephala (Lam.) de

Wit