PHYTOCHEMICAL

ANALYSIS OF
PLANT EXTRACTS
Arizaldo E. Castro
Lecturer I, BSD

Objectives
Orient oneself with the concept of plant extract

preparation.
Carry out phytochemical screening of plant extracts for

compounds such as as:

Alkaloids
Cardiac glycosides
Saponins

Introduction
Phytochemical analysis in Ethnobotany is a relevant

exercise to be completed.
Medicinal plants have been used by rural dwellers

worldwide since antiquity.

Therapeutic potentials of medicinal plants = Diversity of

Secondary metabolites they produce.

 Medicinal and therapeutic applications of plants include

the following
Antioxidants
Antibacterial
Antiviral
Antiparasitic
Anti-inflammatory
Antineoplastic.

 Tests for the presence of: alkaloids, steroids,

terpenoids, anthraquinones, flavonoids, saponins,
tannins, polyphenols

Phytochemical
Sreening

Plant Material

Identity of plant must be established and beyond

question
Voucher specimen must be kept

Plant Extract

Solvent Extraction, Superficial Fluid Extraction,

Microwave-Assisted Extraction, Solid Phase
Extraction

1o vs. 2o Plant Metabolites

Which is produced in bulk, primary or secondary
metabolites?
Primary Metabolites include Carbohydrates, Proteins, and
Lipids
Secondary Metabolites include Alkaloids, Steroids, and
Flavonoids

PHYTOCHEMICALS
Alkaloids
Glycosides
Saponins
Flavonoids
Tannins
Terpenes
Anthraquinones

Alkaloids
Largest group of 2o metabolites
Chemically made up of ammonia compounds (nitrogen bases)

with various radicals replacing one or more of the hydrogen

atoms in the peptide ring, most containing oxygen.
PROPERTIES OF ALKALOIDS
Alkaline in reaction (turns red litmus paper to blue, can be attributed

nitrogen atoms)
Reacts with acids to form crystal salt without the production of water
Most exist as solids
Some exist as liquids
Readily soluble in alcohol
Bitter taste
Used by plants as defense against herbivory and other pathogens

 Uses of alkaloids narcotics, stimulants, poisons

e.g.
Morphine
Codeine
Caffeine
Berberine
Sanguinarine

Glycosides
Condensation products of polysaccharides with different organic

hydroxy compounds, in such a manner that the hemiacetal entity

of the CHO- must essentially take part in the condensation.
PROPERTIES OF GLYCOSIDES
Colorless
Crystalline compounds containing CHO
With water-soluble phytoconstituents
Contain carbohydrate and non-carbohydrate parts (genin or
aglycone).
Neutral in reaction
Can be readily hydrolyzed into its components
Intensely bitter taste

e.g.
Alpha-terpineol
Cinnamyl acetate
Eugenol Taxifolin
Beta-glucosid

Saponins
Possess soaplike behavior in water. They possess distinct
foaming characteristics. They consist of a polycyclic
aglycone that is either a choline steroid or triterpenoid
attached via C3 and an ether bond to a sugar side chain.
The aglycone is referred to as the sapogenin. The ability of
a saponin to foam is caused by the combination of the
nonpolar sapogenin and the water soluble side chain.

PROPERTIES OF SAPONINS
Water soluble
Insoluble in ether
They give aglycones as byproducts after hydrolysis
Extremely poisonous. They can induce hemolysis
Bitter and acrid taste
Can cause irritation to mucuous membranes
Amorphous

 Uses of Saponins: Hypolipidemic, Anticancer actvity

e.g.
Solanine

Procedure
Simple Plant Extraction: Leaves, Barks/Stems, and
Roots (Aguinaldo et al 2005)
Choose your target plant specimen for phytochemical

extraction and screening. Collect approximately 100 g of

leaves, 100 g barks/stems, and 100 g roots of your plant
of choice.
Soak the plant parts in 80% Ethanol for 48 hours inside an

Erlenmeyer flask.

 Filter your sample using Whatmann No. 42 and a Buchner

funnel with gentle suction.

Rinse the flask and plant material with fresh portions of

alcohol. Transfer washings and plant material to the

funnel, combining the washings with the first filtrate
collected.
Discard the plant residue.

 Concentrate the filtrate under vacuo at temperatures

below 50oC to about 20 ml. Measure the exact volume of

extract obtained. Record the concentration of plant extract
as grams of dried plant material per ml of the extract
obtained. Weight the plant extract if necessary to obtain
grams extract per ml of the extract.
Label the plant extract with the name of the plant, the

concentration acquired, and the date of extraction.

Store plant extract in amber bottles at cold, 0-5 oC.

Phytochemical Analysis (Aguinaldo et al 2005)

B.1 Preliminary Analysis for Alkaloid content
Use 20 g plant material equivalent from the plant extract prepared in

procedure A.
Evaporate the extract until syrupy in consistency using a steam bath

set-up.
Add 5 ml of 2 M HCl to the extract and continue heating while stirring

for about 5 min. Cool the sample afterwards.

Add 0.5 g of Sodium Chloride, stir, and filter.

 Wash the residue with enough 2 M HCl to reconstitute a volume

of 5 ml.
Get 1 ml of extract, add 2 to 3 drops of Dragendorffs reagent.

Record your observations.

Get 1 ml of extract, add 2 to 3 drops of Mayers reagent. Record

your observations.
(+) Slight Turbidity
(++) Definite Turbidity
(+++) Heavy Precipitation

 B.2 Analysis for Cardiac glycoside contents

Take an equivalent of 10 g plant material from the stock solution prepared in

Procedure A.
Evaporate sample to incipient dryness over a water bath. Cool to room

temperature.
Defat by taking up the residue with 6 ml of hexane and water, 2:1 v/v.
Partition by gently shaking the mixture in a test tube.
Pipette out the upper hexane layer.
Repeat the treatment with hexane until most of the colored pigments have been
removed. Discard all the hexane extracts properly.
Heat the defatted aqueous layer over a water bath to remove the residual

hexane. Cool to room temperature.

 B.2.1 Keller-Kiliani Test

Add 3 ml ferric chloride to one of the portions obtained in

procedure B.2. Stir.

With the test tube in an inclined position, add 1 ml of

concentrated sulfuric acid allowing it to trickle on the

insides of the test tube.
Allow the mixture to stand upright and observe for any

coloration at the interface of the acid and the aqueous

layers.

 B.3 Analysis for Saponin Contents

Load a capillary tube (15 mm x 1 mm) with the plant extract

obtained from procedure A. Obtain plant extract with a height of

10 mm.
Load another capillary tube with same volume of distilled

water.
Lift the capillary tubes and keep both in a vertical position to

allow the liquid inside to flow out freely.

Compare the height of the liquids in the two tubes.

Thank You