RESEARCH PLAN

Cytotoxicity and Antioxidant Activity of Identified Secondary

Metabolites in Myristica simiarum Leaves

Researchers
Soren David L. Dacles
Christian Josef F. Avelino
Lorraine Zeta M. Diaz

Life Science

A. Rationale

Heart diseases, diabetes, malignant neoplasm and cancer are one of the leading

causes of death in the Philippines (Department of Health, 2019). Patients with low socio-

economic status tend to neglect such diseases due to expensive disease management and

treatment in the Philippines thus, relying on herbal remedies instead of paying expensive

hospital fees. More Filipinos tend to rely on natural products as prevention to various kinds

of diseases. Overproduction of oxidants in the human body is responsible for the

pathogenesis of some diseases. The scavenging of these oxidants is thought to be an

effective measure to depress the level of oxidative stress of organisms. Because of

associated side effects and toxicity of synthetic chemotherapeutic drugs, it would be

important to discover new biologic agents that are less toxic for normal cells that possess

potential antiproliferative effects on cancer cells. Screening active compounds from plants,

leads to the discovery of new medicinal drugs that are promising for the treatment of

various diseases including cancer (Graham et al., 2000).

 Plants are an important source of bioactive compounds with antioxidant capacity

and during the last years, the interest for the use of natural products has significantly

increased. The protective effects of plant secondary metabolites can be attributed to direct

scavenging activities against reactive oxygen species, as well as to the induction of

intracellular antioxidant effect (Zhang et al., 2015). Antioxidant phytochemicals play a key

role in oxidative stress control and in the prevention of related disorders, such as premature

aging, degenerative diseases, diabetes, and cancer (Faraone, Rai, & Chiummiento et al.,

2018). Recent studies also show that phytochemicals in plants exhibit cytotoxicity against

human carcinomas (Aliomrani, Jafarian, & Zolfaghari, 2017).

In the province of Capiz, Tanghas, identified as Myristica simiarum, an evergreen

plant usually found in forests. Often neglected by locals because of its unknown use but

several sources claim that it does have medicinal applications. It is believed that its plant

parts are used to cure skin diseases. Literature review and studies are yet to be examined

and almost none is known on its phytochemical properties and potential therapeutic use,

thus the researchers aim to identify its phytochemical components, to evaluate the cytotoxic

and antioxidant activities.

B. Research Questions, Hypotheses, Scientifc Goals, Expected Outcomes

Research Question

1. What are the phytochemicals present in Myristica simiarum Leaves?

2. What is the effect of the different concentrations of Myristica simiarum Leaves

extract against brine shrimp?

3. What is the effect of the different concentrations of Myristica simiarum Leaves

extract on the DPPH free radical?

Hypotheses

Alternative Hypotheses

1. There is a presence of phytochemicals found in Myristica simiarum leaves.

2. There is a significant difference in the percent mortality of the different

concentrations of Myristica simiarum leaves extract against Brine Shrimp

nauplii.

3. There is a significant difference in the percent inhibition of the different

concentrations of Myristica simiarum leaves extract on the DPPH free

radical.

Null Hypotheses

1. There is no presence of phytochemicals found in Tanghas Myristica

simiarum Leaves.

2. There is no significant difference in the percent mortality of the different

concentrations of Myristica simiarum leaves extract against brine shrimp.

3. There is no significant difference in the percent inhibition of the different

concentrations of Myristica simiarum leaves extract on the DPPH free

radical.
Scientific Goals

The expected results will greatly benefit patients diagnosed with cancer and those

who prevent future health complications. The study will also promote the use of Myristica

simiarum leaves, as a potent antioxidative stress remedy which is natural and locally

available in the community.

The Department of Health (DOH) will benefit from this study since it would mean

another breakthrough and a new discovery in preventing antioxidative stress drugs and

potential anticancer agents thus, reducing the probability of the population diagnosed with

cancer.

Patients diagnosed with cancer with low socio-economic status will benefit from

this study since the product would be less expensive as compared to commercial drugs.

Future researchers who wish to conduct and promote wellness would benefit from

this study consequently persisting in the development of alternative preventive measures

against cancer.

Goals/Expected Outcomes

 To determine if there is a presence of phytochemicals found in Myristica simiarum

Leaves.

 To determine if there is a significant difference in the percent mortality of the

different concentrations of Myristica simiarum leaves extract against Brine

Shrimps.

 To determine if there is a significant difference in the percent inhibition of the

different concentrations of Myristica simiarum leaves extract on the DPPH free

radical.
C. Procedure

The following research institutions will be the venue of the different tests to be

conducted in the study.

 West Visayas State University, La Paz, Iloilo City, will be the site where the

researchers will be conducting the phytochemical screening and Antioxidant

assays.

Materials

The materials to be used in the study will be 500g Myristica simiarum leaves, DPPH

powder, Brine Shrimp eggs, Ascorbic Acid Standard, Hydrochloric acid, Mayer’s reagent,

5% FeCl, 2N NaOH, 10% FeCl, Distilled Water, HNO3, Ninhydrin Reagent, Sulphuric

Acid, Barfoed Reagent, and Molisch’s Reagent.

Tools

The tools to be used in this study will be UV-Visible Spectrophotometer, Cuvettes,

Rotary Evaporator, Test tubes, 100mL volumetric flask, 50mL volumetric flask,

micropipette, Rectangular Glass Container, 100mL Graduated cylinder, Analytical

Balance, Pasteur Pipette, Light Source, Microtip pipette, and Magnifying glass.
Experimental Design

Completely Randomized Design will be used in this study. The preliminary

cytoxicity screening will be measured using Brine Shrimp Lethality Assay (BSLA) and

antioxidant activity will be measured using DPPH Radical Scavenging Activity Assay.

There will be four (4) treatments (1 µg/mL, 10 µg/mL, 100 µg/mL, 1000 µg/mL) for the

brine shrimp lethality assay (BSLA). There will be four (4) treatments (5 µg/mL, 10

µg/mL, 25 µg/mL, 50 µg/mL) with a positive control (ascorbic acid) and a negative control

(Distilled water) for DPPH radical scavenging assay. All experimentations will be done in

three replications mean, standard deviation (SD), and the median lethal concentration

(LC50) and median inhibitory concentration (IC50) values will be computed.

Procedural Design

Gathering of Plant Samples

Plant Sample Preparation

Extraction of Plant Samples

Phytochemical Screening

Determination of the different treatments

Brine Shrimp Toxicity Assay DPPH Radical Scavenging Assay

Data Gathering

Proper Disposal of Tools Used

Statistical Analysis
General Procedure

Gathering of Plant Samples

The leaves of Myristica simiarum will be randomly collected from Poblacion

Takas, Cuartero, Capiz. The plant samples collected will brought to the Office of the

Provincial Agriculturist to be identified and authenticated by Dr. Audie B. Belargo, Senior

Agriculturist.

Plant Sample Preparation

The plant samples will be washed thoroughly to remove extraneous matter. Plant

dehydration method to be used will the method as described by Romero-Buenavides et al.

(2018). Briefly, the collected leaves will be subjected to a dehydration process in a drying

tray with airflow at a temperature of 32°C for seven days.

Extraction of Plant Sample

Maceration method of extraction described by Guevara et al. (2005) will be used.

Two-hundred fifty grams of dried plant samples will be cut and placed in an Erlenmeyer

flask and treated with sufficient 95% ethanol to completely submerge the material. The

flasks will be stoppered and the plant samples will be kept soaked for 48 hours. The filtrate

of the plant sample will be decanted while the plant residue was discarded. The filtrate will

be concentrated under a vacuum using rotary evaporator at a temperature below 50o C. The

extracts will be stored in wide mouth container and will be kept refrigerated until the actual

biological assay. The extracted sample will produce a solid paste, it will be then isolated

and placed in a sterile container.

Phytochemical Screening

The extracted plant sample will be homogenized in 95% ethanol and will be used

for various phytochemical tests. All determinations will be done in two (2) replications.

Test for Alkaloids

2 mL of concentrated Hydrochloric acid (HCl) will be added to 2 mL plant extract.

Then a few drops of Mayer’s reagent will be added using a microtip pipette. The presence

of green color or white precipitation indicates the presence of alkaloids.

Test for Tannins

1 mL of ferric chloride (5% FeCl) will be added to 1 mL of plant extract. The

formation of dark blue or greenish black color indicates the presence of tannins.

Test for Saponins

2 mL distilled water will be added to 2 mL plant extract. Homogenized using a

vortex and shaken manually for 15 minutes. Formation of 1 cm layer of foam indicates

presence of saponins.

Test for Flavonoids

1 mL of 2N sodium hydroxide (NaOH) will be added to 2 mL plant extract.

Formation of dark yellow color indicates the presence of flavonoids.

Test for Phenols

2mL distilled water followed by few drops of 10% ferric chloride will be added to

1 mL plant extract. Formation of blue/green color indicates the presence of phenols.

Test for Proteins and Amino Acid:

Xanthoproteic Test

2 mL of plant extract and 0.5 mL of concentrated nitric acid will be added by the

side of the test tube. Presence of a dark yellow colour indicates the presence of proteins

and amino acids.

Ninhydrin Test

A few drops of Ninhydrin reagent will be added to the plant extract. The formation

of a violet or purplish coloured ring indicates the presence of amino acids.

Test for sugars:

Molisch’s Test

Molisch’s reagent will be prepared. 2 drops of the reagent will be added to the plant

extract. To this solution, 1 mL of concentrated sulphuric acid will be added to the side of

the inclined test tube. Reddish violet ring at the junction of the two layers indicates the

presence of sugars.

Barfoed’s Test

Barfoed reagent will be prepared by dissolving 13.3 g of crystalline neutral copper

actetate in 200mL of 1% acetic solution. The plant extract will be dissolved in distilled

water and heated with the reagent. A red color precipitate indicates the presence of

monosaccharides.

Determination of the Different Treatments

There will be four (4) concentrations as treatments for the Brine Shrimp Lethality

Assay (BSLA) namely: Treatment A – 1 µg/ mL of plant extract; Treatment B – 10 µg/

mL of plant extract; Treatment C – 100 µg/ mL of plant extract; and Treatment D – 1000
µg/ mL of plant extract. There will be four (4) treatments for DPPH radical scavenging

assay namely: Treatment A – 5 µg/ mL of plant extract; Treatment B – 10 µg/ mL of plant

extract; Treatment C – 25 µg/ mL of plant extract; and Treatment D – 50 µg/ mL of plant

extract

Brine Shrimp Lethality Assay

Brine Shrimp Lethality Assay (BSLA) will be used as a preliminary toxicity

screening of the plant extract as described by Olowa and Oneza (2013).

Brine shrimp eggs will be obtained from the Aqua Laboratory of the Office of the

Provincial Agriculturist. Artificial seawater will be prepared by dissolving 38 g of sea salt

in 1 liter of distilled water for hatching the shrimp eggs. The seawater will be placed in a

small plastic container with a partition for dark and light areas. Shrimp eggs will be added

into the dark side of the chamber while a light source above the other side will attract the

hatched shrimp. Two days will be allowed for the shrimp to hatch and mature as nauplii

(larva). After two days, when the shrimp larvae are ready, 4 mL of the artificial seawater

will be added to each test tube and 10 brine shrimps will be introduced into each tube.

Thus, there will be a total of 30 shrimps per concentration. Then the volume will be

adjusted with artificial seawater up to 5 mL per test tube. The test tubes will be left

uncovered under the lamp. The number of surviving shrimps will be counted using a

magnifying glass and recorded after 24 hours. A brine shrimp will be considered dead if it

is immotile and settles at the bottom of the test tube. The percentage mortality will be

computed using the formula:

Number of Nauplii Dead

% Lethality = x100
Total Number of Nauplii
 This is to ensure that the death (mortality) of the nauplii is attributed to the bioactive

compounds present in the plant extracts.

Using probit analysis, the lethality concentration (LC50) will be assessed at 95%

confidence intervals. LC50 of less than 1000 µg/mL will be considered potent. LC50 value

of less than 1000 µg/mL is toxic while LC50 value of greater than 1000 µg/mL is non-

toxic.

DPPH Radical Scavenging Activity

The antioxidant activity was measured using DPPH free radical scavenging assay

as described by Saha et al. (2008) with slight modifications.

Reagent Preparation for DPPH Assay

The DPPH stock solution and plant extracts will be prepared in varying

concentrations. Dilution method will be used in formulating different concentrations from

the stock solution by the formula:

C1 V1 =C2 V2

Where, C1 = Initial Concentration of the Stock solution; V1 = Initial Volume of

solution needed; C2= Final Concentration (Treatment); V2 = Final Volume of the solution.

0.1 mM 1, 1- diphenyl-2- picrylhydrazyl (DPPH)-Ethanol Solution

The 0.00394 grams of DPPH will be dissolved in 10 mL 95% ethanol. The 1 mL

from stock solution will be measured and added to ethanol to come up with 10 mL solution.

Treatment A: 5 ug/mL Extract

In order to make 100 mL of solution, 10 mL from the stock solution of 50ug/mL

will be measured and will be added to 90 mL of distilled water to obtain 100 mL.
Treatment B: 10 ug/mL Extract

In order to make 100 mL of solution, 20 mL from the stock solution of 50 ug/mL

will be measured and will be added to 80 mL of distilled water to obtain 100 mL.

Treatment C: 25 ug/mL Extract

In order to make 100 mL of solution, 50 mL from the stock solution of 50ug/mL

will be measured and will be added to 50 mL of distilled water to obtain 100 mL.

Treatment D: 50 ug/mL Extract

The 0.1 g of extract will be diluted on 2 L of distilled water.

DPPH Assay

The 0.1 mmol/L solution of DPPH in ethanol will be prepared and 1mL of this

solution will be added to 3mL of extract's solution at different concentrations (5, 10, 25

and 50 μg/mL). After 30 min, absorbance will be measured at 517nm using

spectrophotometer.

Vitamin C (ascorbic acid) will be used as a reference drug following the same

concentration of the plant extracts for comparison of the extent of free radical scavenging

activity of the plant extracts. The percentage inhibition activity will be calculated from:

A0 - A1
% inhibition= × 100
A0

Where A0 is the absorbance of the control (DPPH), and A1 is the absorbance of the

extract/ standard. Median inhibitory concentration (IC50) value will be calculated from the

equation of line obtained by plotting a graph of concentration (μg/ml) versus % inhibition.

Data Gathering

The phytochemical analyses, percent lethality, and percent inhibition will be

computed and the corresponding LC50 and lC50 values of the bioassays.
Proper Disposal of Tools Used

Tools used will be washed with a commercial disinfectant and some will be

disposed properly in assigned bins.

Statistical analysis

The data will be analyzed on Statistical Tool for Agricultural Research (STAR)

released by the International Rice Research Institute (IRRI). One-way ANOVA will be

used as test statistic at 1% level of significance.

D. RISK AND SAFETY

 Laboratory gowns, masks, gloves and proper clothes are necessary during the

conduct of the assays.

 Gloves must be worn as protection from hazardous chemicals.

 After removal, wash your hands and gloves should be disposed to avoid

contamination. It should not be used again.

 All hazardous chemicals that are no longer of use will be thrown following the

proper system of waste disposal.

 DPPH solution will be mixed with activated charcoal. It will be transferred in a

chemical waste container and it will be buried in an empty lot to avoid hazard

infliction.

 Laboratory apparatus will be autoclaved for sterilization.

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 Abstract

Due to low socio-economic classes, disease management and treatment in the Philippines
is limited. Use of phytochemicals as an alternative supplement in preventing oxidative
stress, proven effective especially in cancer prevention. An evergreen plant also found in
Western Visayas ⁠— Tanghas (Myristica simiarum) is often neglected but various sources
claim it to have medicinal applications however, almost none is known on its
phytochemical constituents and potential therapeutic use. This study identified the
phytochemicals present in Myristica simiarum leaves, evaluated the cytotoxic, and
antioxidant activity with LC50 and IC50 values, respectively. Myristica simiarum leaves
were randomly collected, dehydrated, homogenized in ethanol and extracted using rotary
evaporation which produced solid pastes and utilized for various assays. The
phytochemicals present in Myristica simiarum leaves were Tannins, Flavonoids, and
Phenols. BSLA was used to determine cytotoxic activity with 96.7 percent lethality with
low LC50 value of 94.39 µg/mL. High Antioxidant Activity using DPPH Radical
Scavenging Activity with 86.79 percent Inhibition with an IC50 value of 19.47 µg/mL.
There are phytochemicals found in Myristica simiarum leaves which influenced the
cytotoxic and antioxidant activity. Results show that Myristica simiarum leaves have the
phytochemicals that can potentially mediate cancer management and prevent oxidative
stress.