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An Investigatory Project Presented to:

Mr. Carlo Stephen Moneva

Department of Biological Sciences

CSM, MSU-IIT

IN PARTIAL FULFILLMENT OF THE PROJECT FOR

BIOLOGY 101.1 (INTRODUCTION TO BIOLOGICAL SCIENCE LABORATORY)

SECOND SEMESTER, SY 2012-2013

Researchers:

Jemateo B. Neri
DIANA ROSE A. FERNANDEZ

NADJIFAH S. BAYAO

ZENYFAYE G. SALIG

MARCHELLE B. PAGALARAN
 INTRODUCTION

The earth is wealthy of natural and unique medicinal plants. Early in human

history, people practiced herbal medicine as a magical or religious healing art. Herbal

remedies have been used for thousands of years. In the rest of the world, approximately

64 percent of the population relies on herbal medicines. Despite their overwhelming

popularity and long history, we know relatively little about the safety and effectiveness of

herbal remedies. Global recognition of natures green pharmacy should inspire

individuals and nations to protect this extraordinary resource (Pelletier, 2009).

MODERN PHARMACEUTICALS CANNOT TREAT EVERY CONDITION EFFECTIVELY, AND SOME

DRUGS HAVE UNWANTED SIDE EFFECTS. THUS, MANY RESEARCHERS HAVE STUDIED SUCH
ALTERNATIVE MEDICINES. A WAY OF DISCOVERING THE MEDICINAL COMPONENTS IS TO
INVESTIGATE THE CERTAIN PHYTOCHEMICALS LIKE , FLAVONOID, CARDENOLIDES, TANNINS AND
PHENOL.

In this study, the researchers would like to investigate the phytochemicals

present in the Calabash tree leaves and determine if the said plant can be used as an

alternative medicine.

Background of Study

Calabash is identified in different places for the reason of its essential elements

especially to its fruit. Calabash tree or Crescentia cujete tree belongs to the family of

Binoniacea. It is also known as the gourd tree (Gilman, 1993).

Practically, all parts of the tree have been found to be useful. The wood is used
for tool handles, ribs in boat building and cattle yokes; and the gourd for cups,

containers and musical instruments (Burkill, et al., 1985). According to Michael, et al.,

(2004), the fruit was reported to have medicinal application. The extract of the fruit is

useful in the treatment of fever and the plant is used most frequently for the effective

treatment of diseases of the respiratory tract such as: bronchitis, whooping cough,

asthma and those related to illnesses caused by the cold (Biotrade, 2005).

(TKasutlat yqt TyStTebqbsbuSCrescentia CujeteT-ecawyTibuTuyaiwtiTescTwyuTeSt webqTes uywyat yuT)

RseSt webqTxcsxtcywtuxcspw bytTb iT w tcbqTes xsuwyws TbuTitqqTbuTxSty presence of
phytochemicals like saponins, flavonoid, cardenolides, tannins and phenol as well as
the presence of hydrogen cyanide were observed in the fruit sample (Ejelonu, et al.,
2011T wstiTysTstTb yw bqbcwbqTb iTbeyTbuTb bqTtuweEStutTxStyseSt webquTqwctTbqcbqswiuTbctTctesTTR)
anthraquinones as antioxidantw eqb bysct Tyb w uTbctTc si TbuTb ywiwbccStb T-ubxs w uTbuTb ywT
Reb etcTb iTb yw wecsswbq-b iTeqb s swiuTbuTysTstTb yw

Thus, the researchers aim to investigate the phytochemicals present in the

leaves of Crescentia cujete, commonly known as calabash tree, and its significance as

an alternative herbal treatment.

Objectives of the Study

This study aims to determine the phytochemicals present in the extract of the

Calabash (Crescentia cujete) leaves. Specifically, its objective is; to conduct qualitative

screening for phytochemicals such as tannins, saponins, alkaloids, flavonoids and

anthraquinones. The health benefits of the photochemical present in the calabash fruit

extract.

Significance of the Study

Nowadays, many people are suffering from different illnesses and diseases in

our society and they are searching for guaranteed alternative medicines by the use of
plants and plant products. One assurance to recognize the effectiveness of a certain

herbal medicine is to prove whether it has advantageous therapeutic relief or other

necessary procedures.

This study is very helpful especially to those people who cannot buy expensive
medicines. It can also teach them on how to prepare some herbal remedies that can be
very useful in their everyday lives in order to lessen the usage of synthetic medicines.

The researchers aimed to know good effects of studying some herbal plants that
can contribute in the society and in the field of Science.

Scopes and Limitations

This study aims to analyze on the leaves of the Calabash tree that were collected

in Barangay Dalipuga, Iligan City. The study only focuses on the phytochemicals that

will be found and other constituents present in the leaves of the tree. Other information

not related is not included in the study.

 METHODOLOGY

I. Materials:

Hydrochloric Acid METHANOL (400ML)

Knife
3 BEAKERS (400ML)
PIECE OF CLOTH
GELATIN
CALCIUM HYDROXIDE SOLUTION
HEXANE (10ML)
5 BEAKERS (250ML)
DISTILLED WATER

1 Beaker (1000ml)
Stirring Rod

CHOPPING BOARD
FILTER PAPER
SODIUM CHLORIDE
FUNNEL RACK
SPOON
FUNNEL
ACETIC AHYDRIDE
SEPARATORY FUNNEL
GRADUATED CYLINDER
AMMONIA (5ML)

Ferric Chloride SAMPLE HOLDER

CALABASH LEAVES
BLENDER

ETHYL ACETATE (5MM) Wire Gauze

II. Extraction of Calabash Leaves:

Collect leaves from the Calabash tree. Wash the leaves and chop it into

small pieces. Blend the Calabash leaves using blender, mix it with 1 cup of water and 1

cup of leaves. Using a strainer or a piece of cotton cloth, drain the blended Calabash

leaves to get the juice and separate it with the residue. After that, leave the unwanted

matter for disposal. Once it cools, put into bottles and refrigerate.

III. Content Determination:

A. Alkaloids

A.1. Preliminary Test

A 100mg of an extract was dissolved in dilute hydrochloric acid. Solution was clarified

by filtration. Filtrate was tested with Dragendorffs and Mayers reagents. The treated

solution was observed for any precipitation.

A.2. Confirmatory Test

Five grams of the extract was treated with 40% Calcium hydroxide solution until the

extract was distinctly alkaline to litmus paper, and then extracted twice with 10 ml

portions of chloroform. Chloroform extracts were combined and concentrated in vacuole

to about 5ml. Chloroform extract was then spotted on thin layer plates. Solvent system

(n-hexane-ethyl acetate, 4:1) was used to develop chromatogram and detected by

spraying the chromatograms with freshly prepared Dragendorffs spray reagent. An

orange or dark colored spots against a pale yellow background was confirmatory
evidence for the presence of alkaloids.

B. Saponins

B.1. THE FROTH TEST

0.5g extracts were dissolved in 10ml of distilled water for about 30 seconds. The test

tube was stoppered and shaken vigorously for about 30 seconds The test tube was

allowed to stand in a vertical position and observed over 30 minutes period of time. If a

honey comb froth above the surface of liquid persists after 30 minutes the sample is

suspected to contain saponin.

B.2. LIEBERMANN BURCHARD TEST

0.5g of the extract will be dissolved in 2ml of acetic anhydride and cooled well in ice
sulphuric acid was then carefully added. A colour change from violet to blue to green
will indicate the presence of a steroidal nucleus.

C. TANNINS

C.1. Ferric Chloride Test

A portion of the extracts was dissolved in water. The solution was clarified by filtration;

10% ferric chloride solution was added to the clear filtrate. This was observed for a

change in colour to bluish black.

D. Flavonoids

D.1. TESTS FOR FREE FLAVANOIDS

5mm of ethyl acetate was added to a solution of 0.5g of the extract in water. The
mixture was shaken, allowed to settle, and inspected for the production of yellow colour

in the organic layer, which is taken as positive for free flavanoids.

D.2. LEAD ACETATE TEST

To a solution of 0.5 g extract in water, about 1ml of 10% lead acetate solution was
added. Production of yellow precipitate is considered as positive for flavanoids.

D.3. Reaction with Sodium hydroxide

Dilute sodium hydroxide solution was added to a solution of 0.5g of the extract in water.

The mixture was inspected for the production of yellow colour which considered as

positive test for flavanoids.

E. Steroids

E.1. Keller Kiliani test

0.5 of extract will be dissolved in 2ml of glacial acetic acid containing one drop of ferric
chloride solution. This will then be underlayed with 1ml of concentrated sulphuric acid.

H. Anthraquinones

H.1. BORNTRAGER'S TEST

5g of each plant extract is to be boiled with 10ml aqueous sulphuric acid and filtered

while hot. The filtrate was shaken with 5ml of benzene, the benzene layer separated

and half its own volume of 10 percent ammonia solution added. A pink or violet

coloration in the ammonia phase (lower layer) indicates the presence of anthroquinones

derivatives in the extract.

 REFERENCES

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chemists. 15th Edn. Washington D.C.
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Burkill HM (1985). The useful plants of Tropical West Africa. 2nd Edn. Richmond, UK,
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THERESA 2011, ESSENTIAL OIL PURITY AND SAFETY TESTING METHODS, JULY 2011,
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