PHYTOCHEMICAL ANALYSIS AND COMPARATIVE TOXICITY TEST (LC50)

OF MALUNGGAY (Moringa oleifera) ETHANOLIC

ROOT AND LEAF EXTRACT

A Research Paper Presented to the Faculty of

AGUSAN NATIONAL HIGH SCHOOL
Senior High School Department
Butuan City

In Partial Fulfilment of the Requirements for the subject

Capstone Research

By

Genie Joy A. Bangui

Ira Karen A. Dispo
Glychell T. Euldan
Fiona Abegaile B. Faelnar
Awie T. Macarik
Loven R. Natulla

JULY 2019
 Chapter 1

THE PROBLEM AND REVIEW OF RELATED LITERATURE

BACKGROUND OF THIS STUDY

Malunggay (Moringa oleifera) is usually comprehended to

be significant on the human health. Moringa oleifera or also

known as Malunggay which is one of the major plants that

will cure severe illnesses, natural coagulant, effective to

treat wounds and commonly a beneficiary to the human health.

This plant is widely been used as food ingredients as it is

widely used to be nutritious like Malunggay soup and many

others. According to Jimenez et. al. (2017), Malunggay is

mostly enrich of significant compounds that are needed to

create helpful products such as medicines.

Although, behind its entire beneficial outcome to

humans, it is still considered to have negative effects on

the fertility of a female that undergoes miscarriage (Viera,

2010). This plant can also affect the human heart rate by

slowing it down and decreases the blood pressure of a human

body (Whitmer, 2019). In addition, there is a part of

Malunggay (Moringa oleifera) like roots that contain a toxic

substance that can cause paralysis or even death. The

purpose of this study is to focus and dig more information

about the toxicity of a Malunggay (Moringa oleifera). This

will contribute instruments to examine the roots and leaves

of the Malunggay (Moringa oleifera) through phytochemical

and toxicity test.

Constantly, on the other hand this remedy doesn’t

always have a good result on the daily health of humans.

Hereby, this study will show the other bad side-effects of

Malunggay (Moringa oleifera), in order for the readers to

gain new information regarding the toxicity of this plant.

Objectives of the Study

In accordance with the needs of the study, the

following specific objectives were determined:

1. To conduct a qualitative phytochemical screening of

the ethanolic extract of Moringa oleifera.

2. To test the toxicity of the ethanolic leaf and root

extract of Moringa oleifera using brine shrimp as

specimen to determine its Median Lethal

Concentration (LC50).

3. To determine if there is a significant difference

between the mortality rates of the brine shrimp when

applied by different ethanolic root extract and leaf

extract concentrations?
Statement of the Problem

On this context, the study will conduct phytochemical

screening to assess the Malunggay (Moringa oleifera)

secondary metabolites and the median lethality concentration

(LC50) toxicity test. Specifically, this study will aim to

answer the following questions:

1. What is the phytochemical screening of the Malunggay

(Moringa oleifera) leaf and root extract?

2. What is the level of toxicity of the different

ethanolic leaf and root extract concentrations?

3. What is the maximum level of toxicity of the

Malunggay (Moringa oleifera) leaf and root extract

in terms of different concentrations?

4. Is there significant difference between the

mortality rate of the brine shrimp when applied by

the different ethanolic root extract concentrations?

5. Is there significant difference between the

mortality rate of the brine shrimp when applied by

the different ethanolic leaf extract concentrations?

6. Is there a significant difference between the levels

of toxicity of roots and leaves?

Statement of the Hypothesis

The roots and leaves of Malunggay (Moringa oleifera)

does not possess phytochemical properties. The maximum level

of toxicity of the Malunggay (Moringa oleifera) of leaf and

root extract in terms of different concentrations is zero.

There is no significant difference between the mortality

rate of the brine shrimp when it is applied by the different

ethanolic leaf and root extract concentrations.

Theoretical Framework

This study focuses on the properties and toxicity of

the plant which is the Malunggay (Moringa oleifera). This

examination discusses about the phytochemical analyzation in

order to know if there is a possible chance of property that

can harm pregnant women and people’s health. It also

discusses about the LC50, which means the standard measure

of the toxicity of the surrounding medium that will kill

half of the sample population of a specific test-animal in a

specified period through exposure via inhalation

(respiration). LC50 will be used if there would be a

possibility of toxicity or none on the Malunggay (Moringa

oleifera).
Conceptual Framework

The independent variables of the study refers to the

Malunggay (Moringa oleifera) root and leaf extract. The

intervening variable will be the amount of extract’s

concentration that will be used to determine the levels of

toxicity of the Malunggay (Moringa oleifera) root extract.

The dependent variable will be the analyzation the

phytochemical of properties, toxicity and the mortality rate

of the amount of the brine shrimp with respond to the

extract’s concentration.

Research Paradigm

Independent Dependent
Variable Variable
Intervening
Malunggay Analyzation
Variable of properties
(Moringa Amount of
oleifera) Extract’s Mortality
Concentration Rate of Brine
Root extract Shrimp
Leaf extract
Figure 1. Research Paradigm Showing the Interplay between
the Independent Variable and Dependent Variables of the
Study.

Scope and Limitation

The researchers will limit the Malunggay (Moringa

oleifera) from Tinago, Matabao, Buenavista, Agusan Del

Norte. Maceration and distillation process of the following

ethanolic extracts will be done at Department of Science and

Technology (DOST). The study will be conducted under

laboratory conditions. The brand of the brine shrimp is

Artemix. The brine shrimp was purchased online. For the

detection of the LC50, brine shrimp will be induced with

different concentrations of ethanolic extract and a control

will be administered through a trial and error set up at

Caraga State University Biology Laboratory. The obtained

plant sample will be processed for the qualitative

phytochemical analysis at the CSU Biology Laboratory.

Significance of the Study

In this study, the researchers believe that the

following would benefit from the results of the study:

Students. This help the students to know more about the

benefits and the harmfulness that Malunggay (Moringa

oleifera) contains.
 Teachers. This study will encourage teachers to produce

more similar research that is based on their specific needs.

Household. This study will help to raise awareness such

as pregnant women to be aware of the danger of eating the

specific part of the Malunggay (Moringa oleifera) that can

cause miscarriage and abortion.

Community. This study would help the community to raise

awareness of the consumption of Malunggay (Moringa

oleifera).

Future Researchers. This study will help those

researchers to continue more discussions and discoveries to

this type of field of study. This help the researchers to

help the community in raising awareness to people.

Definition of Terms

The following terms are operationally defined for the

purpose of clarity.

Ethanolic Extract. It is the process of extracting

essential oils and cannabinoids from the marijuana plant by

using ethyl or isopropyl as the solvent.

Leaf. One of the flat and typically green parts of a

plant that grow from a stem or twig.

 Lethal Concentration 50 (LC50). Standard measure of the

toxicity of the surrounding medium that will kill half of

the sample population of a specific test-animal in a

specified period through exposure via inhalation

(respiration).

Moringa oleifera. It is a plant that is native to sub-

Himalayan areas of India, Pakistan, Bangladesh, and

Afghanistan. The leaves, bark, flowers, fruit, seeds, and

root are used to make medicine.

Phytochemical Analysis. It refers to the extraction,

screening and identification of the medicinally active

substances found in plants.

Root. The part of a plant that grows underground, gets

water from the ground, and holds the plant in place.

Review of Related Literature

There have been a number of plants that are constantly

being screened for their phytochemical value because natural

drugs can be obtained from the plants.

Family and Structure of Malunggay (Moringa oleifera)

There are many different types of plant in the world.

Plants are important as a source of food, energy,

traditional medicine and shelter for both human beings and

animal. There are many useful harvests that are obtained

from different kind of plants directly or indirectly

claiming their importance to the human being and other

living organisms in the entire world.

The “Moringa oleifera” belongs to the 13 species of

family Moringaceae. It is native to India, Africa, South

America, Arabia, South Asia and the Pacific and also the

Caribbean Islands. Moringa oleifera has been developed in

many countries in the tropic and subtropics regions

worldwide (Mustapha, 2013.)

The plant has a lot of names that can be called a

drumstick tree, horseradish tree, miracle tree, or “Mothers

best friend”. Moringa oleifera is widely known as

“Drumstick” (Ijeoma, 2012.) It can be found in the sub-

Himalayan tract and it is either small or medium sized tree,

with a 10m height. The Moringa oleifera is a fast-growing

tree or deciduous tree. The Moringa usually grows up to 10

to 12m in its height. It has the structure of an open crown

of drooping fragile branches, whitish bark, feathery foliage

of tripinnate leaves and thick corky. Moringa grows rapidly,

reaching higher heights. It can be found in well-drained

soils with ample water but it also tolerates sandy soils and

heavier clay soils and water-limited conditions. Its leaves

contain phytochemical having potent hypotensive activity and

anticancer and considered full of medicinal properties and

is used in Siddha medicine (Sharma et.al, 2010).

Benefits of Malunggay (Moringa oleifera)

Moringa oleifera has been used as an herbal and

traditional medicine in different cultures and different

countries for so many years. It is known as a nutritious

plant that is widely used for fighting malnutrition in the

world and mostly used for infants and nursing mother.

According to Ekor (2014), about 70–80 % of the world’s

population especially in developing countries relies on

herbal medicine to treat and prevent diseases. About 25 % of

the processed drugs are manufactured from medicinal plants

(Pan et al. 2013).

Moringa oleifera is used as a drug in the treatment of

asthma and possess the antithyroid, antiasthmatic,

antidiabetic, antiepileptic, antimicrobial, antibacterial,

antibiotic, anti-inflammatory, antioxidant, antiulcer,

antispasmodic, cholesterol-lowering, anti-HSV, antifungal,

diuretic, antihypertensive, hepatoprotective, antitumor

activity (Gupta et.al, 2013.)

The pods of the plant are the most valued and widely

used part of the plant because it contains essential

vitamins, amino acids and other nutrients. The pods may be

eaten cooked or raw. When the pods are fried, it may produce

an odourless, clear and sweet oil that is usually called the

“Ben Oil”.

The leaves of the Moringa oleifera can be eaten as

greens, it can be mixed in the salads or it can be a

vegetable ingredient for soups and other tropical

delicacies. The leaves of Moringa oleifera are the most

widely studied in the world because it shows to have

benefited in several conditions such as high blood pressure,

chronic conditions, hypercholesterolemia, diabetes, insulin

resistance, also liver disease, cancer and overall

inflammation.

The flower can be cooked and can be eaten either mixed

with other foods or fried in batter. Traditionally its roots

are applied as a plaster to reduce the swelling and

rheumatism. The root, flower, fruit and leaf have analgesic

and anti-inflammatory activity.

According to Padayache and Bajinath (2012), the Moringa

is often considered as an important famine food because it

has a high resistance to arid conditions owing to their

tuberous roots and also to drought. The Moringa tree is

useful especially for medicinal, functional preparation,

nutraceuticals, water purification and biodiesel production;

including roots, leaves, flowers, green pods and seeds

(Saini, 2015).

Malunggay is also used to boost and increase the immune

system, sex drive (as an aphrodisiac), reduce swelling, and

increase breast milk production. Some people use it as a

nutritional tonic or supplement. Acording to Sayeed et.al

(2012), Malunggay is sometimes used and applied directly to

the skin as a drying agent or germ-killer (astringent). It

is also used and applied typically for curing pockets of

infection (abscesses), dandruff, snake bites, warts, wounds,

athlete’s foot and gum disease (gingivitis).

All parts of the Moringa oleifera plant is used and

applied in the treatment of fever, psychosis, eye diseases

and as an aphrodisiac. The roots and barks are found to be

effective for preventing implantation because of its aqueous

extracts, the fruits have shown importance of anti-

inflammatory activity because of its aqueous extracts, and

also the leaves have found to have an antiulcer activity due

to its methanolic extracts, also the seeds exhibited

antitumour activity due to its ethanolic (Rameshwar, e.t al,

2010.)

Active Compounds of the Moringa oleifera

Moringa oleifera consists of active components that are

important properties inside the plant. It is been widely

beneficiaries in several illness conditions including

hypocholesteremia, high blood pressure, insulin resistance,

diabetes, cancer, non-alcoholic liver disease, and overall

inflammation. According to the article “Bioactive Components

in Moringa oleifera Leaves Protect against Chronic Disease”

by (Jiminez et. Al, 2017), they showed compounds significant

related to Moringa oleifera may include:

Vitamins

Moringa oleifera leaves are a source in carotenoids of

pro-vitamin A potential (Slimani, 2011.) and a source of

vitamin A (Ferreira, 2010). Its leaves consist of 200

milligrams per 100 grams of vitamin C, and the concentration

is above than oranges (Ramachandran, 2012). Moringa

oleifera leaves are commonly known to be useful in the

embryonic process, reproduction, vision, and development,

cell contrast and immune competence (Alvarez, 2014). Vitamin

E is important because it acts as antioxidants, but it also

adapts to the proliferation of the cell and it has the same

concentration to nuts (Efiong, 2013).

Polyphenols

Moringa oleifera is consisting of polyphenol properties

involving phenolic and flavonoids acids. Flavonoid acids

will control the microbial to prevent harmful disease and it

has a benzo y-pyrone ring as a common structure (Kumar,

2013). It will assist to protect the human body like

oxidative stress, including cancer and cardiovascular

disease. MO is a source of flavonoids (Pandey, 2013) and it

has main flavonoids such as also kaempferol, quercetin, and

myricetin, in concentrations of 5.8, 0.207 and 7.57

milligram per gram (Coppin, 2013). Quercetin is found in

flavonoids that use as antioxidants, with different

compounds of therapeutic (Bischoff, 2011).

Phenolic acids are compounds derived from hydroxycinnamic

acid and hydroxybenzoic acid. It is commonly used as anti of

mutagenic, cancer, and inflammation properties (El-seedi,

2012). Gallic acid is the most abundant in dried leaves with

a concentration of 1.034 milligrams per gram of dry weight.

Chlorogenic acid is a top phenolic acid in MO and acts as a

metabolism of glucose (Karthikesan, 2012).

Alkaloids, Glucosinolates, and Isothiocyanates

Alkaloids is a faction of chemical properties, which

consist commonly of atoms of nitrogen. It contains compounds

like phenylacetonitrile, pyrrolemarumine, rhamnopyranose

lincosamide, glucopyranosyl, and 4’-hyrdroxyphenylethanamide

rhamnopyranoside, have been contained in Moringa oleifera

leaves (Panda, 2014).

Glucosinates is the secondary faction of metabolites in

every plant. Glucosinates and isothiocyanates have been role

to make a significant role in promoting the health compounds

of a plant (Dinkova, 2012).

Toxicity

The Moringa oleifera is safe when it is taken by mouth

and used appropriately. The seed, fruit, and leaves are safe

when eaten as food. Although it is important to avoid eating

its extract and the roots. The parts of the Moringa

oleifera may contain a toxic substance that can cause

paralysis or even death. Moringa oleifera has been used

safely in doses up to 6 grams daily for up to 3 weeks.

According to Viera e.t.al (2010), being pregnant is

likely unsafe to use the bark, flower or the root of the

Moringa oleifera. The chemicals in the bark, flower or the

root can make the uterus contract and can cause miscarriage.

The Aqueous extract of Moringa oleifera roots was found to

be effective as anti-fertility in the absence or the

presence of the progesterone and estradiol dipropionate. In

the early stages, it is traditionally used to abort

pregnancies in the early stages. The roots contain

spirochin, a toxic substance. Pregnant women should avoid

eating the roots, leaves and flower of the Moringa oleifera

plant. The roots trigger many abortions and reduced

fertility in animals

The Moringa oleifera has a property of alkaloids in the

plant that may slow the heart rate and decrease blood
pressure. It also has an isolation of chemical from roasted

Moringa oleifera seeds that causes cell mutations. Moringa

oleifera leaves can also increase the risk of kidney and

liver damage in rats (Whitmer, 2019.)

Chapter 2
METHODOLOGY

Research Design

This study uses two group design, this design is the

most appropriate because there are two variable used in the

study. Specifically, Malunggay (Moringa oleifera) extract

will serve as the major variable and its toxicity will be

tested through the LC50 with different amounts of

concentration. A two-group design is when a researcher

divides his or her subjects into two groups and then

compares the results (Toulany, 2013).

Statistical Treatment
 The statistical tools to be used in analysing,

interpreting of data, and testing the null hypothesis of the

proposed study will include the frequency counts, Motality

rate , and t-test.

Mortality Rate will be used to determine the toxicity

of Malunggay through getting the average of the brine shrimp

mortality. Mortality rate is defined as to measure the

frequency of occurrence of death in a defined population

during a specified interval. The result will be the number

of deaths in a given time period over the population from

which the deaths occurred in the same time period and

multiplied it by 100 (Mausner and Kramer, 2010, pg.101). The

formula of the mortality rate is:

MORTALITY RATE= number of deaths in a given time period

X100
the population from which the deaths occurred in the same
time period

A t-test is to determine the likelihood of a value in a

sample, given that the null hypothesis is true. A t-test

(also known as Student's t-test) is often used to test if

two samples are statistically different from each other.

A t-test (also known as Student's t-test) is often used

to test if two samples are statistically different from each

other. A t-test does this by comparing the means of both

samples. The formula for the t-test is a ratio. The top part

Mortality rate of Brine Shrimp

Concentration
Replicate Replicate Replicate
No.1 No.2 No. 3
T1

T2

T3

T4

T5

of the ratio is just the difference between the two means or

averages. The bottom part is a measure of the variability or

dispersion of the scores.

The level of significance that will be employed in this

study is through the alpha level of 0.05 or 95% level of

significance to determine the significance of the findings.

Table 2. Number of percentage mortality of the toxicity test

of Malunggay (Moringa oleifera) leaf extract.
.

Table 2. Number of percentage mortality of the toxicity test

of Malunggay (Moringa oleifera) root extract.
 Mortality rate of Brine Shrimp
Concentration
Replicate Replicate Replicate
No.1 No. 2 No.3
T1
T2
T3
T4
T5

Procedure

Preparation of Malunggay (Moringa oleifera)

Preparation of Moringa oleifera ethanolic extracts are

the roots that will be collected from Tinago, Matabao,

Buenavista, Agusan del Norte. The leaves and roots will be

rinsed and air dried for 3 days, it will be then sent to

Department of Science and Technology for the extraction

proper.

Extraction of Malunggay (Moringa oleifera)

The Malunggay (Moringa oleifera) were extracted by

Department of Science and Technology (DOST) by using rotary

vapor to remove solvents from the roots and leaves of the

Malunggay (Moringa oleifera) by under reduced pressure.

Rotary vapor is a piece of apparatus consisting of a motor

unit that rotates the evaporation flask, a vacuum system, a

heated water bath and a condenser, which is used to remove

solvents from samples under reduced pressure (Jessop et.al.,

2010).
Test for Phytochemical Analysis adapted from Tiwari.

Test for alkaloids

0.2 mg of each plant extract will be placed in

separate test tubes and warmed with 2mL of 2% sulphuric acid

for two minutes. It will be then filtered in separate test

tubes and 3 drops of Dragendorff‘s reagent will be added.

The appearance of orange-red precipitates will be an

indication for the presence of alkaloids.

Test for Terpenoids`

0.5g of each plant extract will be placed in separate

test tubes containing 2 mL of chloroform. 5 drops of

concentrated sulphuric acid will be carefully added to form

a layer. The presence of red-brown color interface will be

observed to validate its existence.

Test for Reducing Sugar

Three test tubes with 2 ml of plant extract in each

separate tube will be added with 5 mL of distilled water and

five to eight drops of Fehling‘s solution. It will be then

heated over a water bath for five minutes. A red brick

precipitates will be an indication for the presence of

reducing sugars.

Test for Saponins

In each test tube, 1 g of each plant material will be

loaded. 10 ml of distilled water will be added and the

mixture will be boiled for five minutes. The mixture will be

then filtered and 2.5 mL of the filtrate will be included in

a test tube with 10 ml of distilled water. The test tube

will be secured with a stopper and will be shaken vigorously

for about thirty seconds and allowed to stand for 30

minutes. Honeycomb froth will be an indication for the

presence of saponins.

Test for Tannins

1gram of each plant extract will be mixed with 5mL of

distilled water and will be heated on a water bath. The

mixture will be then filtered, and a pinch of ferric

chloride will be added. A dark green color will implied the

presence of tannins.

Test for Steroids

2 mL of acetic acid and 0.5 mg of the ethanol extract

of each sample will be added with 2 mL of sulphuric acid.

The change of blue or green from violet will be an

indication of the presence of steroids.

Test for Phlobatannin

0.5 mg of plant extract in each test tube will be

dissolved in 5mL distilled water. It will be then filtered,

and the filtrate will be brought to a boil with 5mL of 2%

HCl solution. The formation of red precipitates will

indicate the presence of Phlobatanin.

Test for Glycosides

A 5 mL of diluted sulphuric acid will be added to the 1

mL extract in test tubes. It will be then boiled for fifteen

minutes and will be cooled and neutralized with 5mL of 10%

NaOH. 1mL of Fehling solution will also be added. The

formation of red brick precipitates will be an indication of

the presence of glycosides.

Test for Volatile Oils

On separate sterile test tube, 2 mL of different plant

extract will be loaded. The extracts will be shaken with 0.1

mL of diluted NaOH and 2mL of diluted HCl. The presence of

white precipitates will be an indication for the presence of

volatile oils.

Toxicity Test

Procurement and Preparation of Brine Shrimp Larvae (Artemia

Cysts)

The brine shrimp that will be used in the study will be

purchased online weighing 20 grams. An improvised brine

shrimp hatching container will comprise1 liter of sterilized

soda bottle. An air pump will be added to the vessel

allowing sufficient oxygen consumption for the larvae to

hatch. 500 ml of water with saline solution will be added to

the vessel together with the 16 grams of brine shrimp

(suggested ratio for utmost hatching). A light bulb will be

subjected to the solution for maintaining a warm temperature

which will be needed for conducive hatching. The hatching

procedure will cover up to 2 days (48 hours), the newly

hatched brine shrimp will be transferred into a petri dish

with cover by using a drinking straw and will be set aside

for the experiment proper.

Administration of the Concentration on each Set-Up

The determination of LC50 for toxicity will be done

through the testing of plant samples in various

concentrations. The dilution will be ten-fold, the following

concentrations are: 10,000ppm, 1,000ppm, 100ppm, 10ppm and

1ppm. On each test tube, 20 pcs of brine shrimp will be

added to initiate the experiment. The same application will

be done for the two plant samples respectively. The LC 50

will be identified and recorded with varying extracts and

concentrations and will be done in a 24-hour exposure.

Data Gathering and Analysis

The lower and upper roots of the Malunggay (Moringa

oleifera) were extracted and has a weight of 1 kilogram. The

brine shrimp were used to test the toxicity of the Malunggay

(Moringa oleifera) by counting the de-funct ones. The

observation were recorded using a camera for the

documentation. The data were recorded as well using a pen

and a paper. The data were then statistically analyzed by

the researchers using Microsoft Excel. One-Way ANOVA was

used to test the mortality rate of the brine shrimp. The

amount of concentration were determined by the percentage of

levels of toxicity.
Flowchart Diagram of the Procedure

Preparation of
Malunggay (Moringa
oleifera)

Extraction of
Malunggay

Test for
Phytochemical
Analysis adapted from
Tiwari

Toxicity Test

Procurement and
Preparation of Brine
Shrimp Larvae
(Artemia Cysts)

Administration of the
Concentration on each
Set-Up
 Data Gathering and
Analysis

Figure 2. Flowchart

Diagram of the procedure showing the step by step process