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PTT 312

Extraction of Bioactive Compound

Extraction and purification

of bioactive compound
 EXTRACTION is the first step of any medicinal plant study, plays a
significant and crucial role on the final result and outcome.

 The development of modern chromatographic and spectrometric

techniques make bioactive compound analysis easier than before but
the success still depends on the extraction methods, input
parameters and exact nature of plant parts

 The most common factors affecting extraction processes are;

- matrix properties of the plant part
- solvent
- temperature
- pressure
- time

 Applications: pharmaceuticals, food additives, cosmetics,

natural pesticides
Plant’s Bioactive compound :

A secondary plant metabolites eliciting pharmacological or

toxicological effects in human and animals


Terpenes & Alkaloids Phenolic

Terpenoids Compounds
(approximately 12,000 types) (approximately
25,000 types), 8000 types).
Alkaloids Nicotine tobacco coca plant interfere with
cocaine chocolate (cocao) neurotransmission, block
enzyme action
Monoterpenes menthol mint and relatives, many interfere with
linalool plants neurotransmission, block ion
transport, anesthetic

Sesquiterpenes parthenolid Parthenium and relatives contact dermatitis

( Asteraceae )
Diterpenes gossypol cotton block phosphorylation; toxic

Triterpenes, cardiac digitogenin Digitalis (foxglove) stimulate heart muscle,

glycosides alter ion transport
Tetraterpenoids carotene many plants antioxidant; orange coloring

Terpene polymers rubber Hevea (rubber) trees, gum up insects; airplane

dandelion tires
Sterols spinasterol spinach interfere with animal
hormone action
Phenolic acids caffeic, chlorogenic all plants cause oxidative damage,
browning in fruits and wine

Coumarins umbelliferone carrots, parsnip cross-link DNA, block cell

Lignans podophyllin may apple poison cathartic, vomiting, allergic
urushiol ivy dermatitis

Flavonoids anthocyanin, almost all plants flower, leaf color; inhibit

catechin enzymes, anti- and pro-
oxidants, estrogenic

Tannins gallotannin, oak, hemlock trees, bind to proteins, enzymes,

condensed tannin birds foot trefoil, block digestion,
legumes antioxidants

Lignin lignin all land plants structure, toughness, fiber

Different extraction techniques should be used in diverse
conditions for understanding the extraction selectivity from
various natural sources.
All these techniques have some common objectives;

(a) to extract targeted bioactive compounds from complex

plant sample
(b) to increase selectivity of analytical methods
(c) to convert the bioactive compounds into a more suitable form
for detection and separation,
(d) to provide a strong and reproducible method that is
independent of variations in the sample matrix
Conventional Extraction Non-conventional Extraction
(1) Soxhlet extraction (1) Ultrasound assisted extraction
(2) Maceration (2) Microwave-assisted extraction
(3) Hydro distillation. (3) Supercritical fluid extraction
(4) Pressurized liquid extraction
(5) Enzyme-assisted extraction

 Longer extraction time  Use safer chemicals/solvent

 Require costly and high  Design for energy efficiency
purity solvent  Design to prevent degradation,
 Use/evaporate huge atom economy, and time analysis
amount of solvent, for pollution prevention and
 Low extraction selectivity inherently safer chemistry for the
 Thermal decomposition of prevention of accident
thermo labile compounds
Soxhlet Extraction
Small amount of dry sample is placed in a thimble

Thimble is then placed in the extraction/Soxhlet

chamber which connected to the flask
containing solvent and condenser at the top

Vapor of heated solvent is condensed and flow

through the sample. After reaching to an overflow
level, the solution inside the extraction chamber is
aspirated by a siphon.

Siphon unloads the solution back into the flask.

This solution carries extracted solutes into the
bulk liquid.

Solute is remained in the flask and solvent passes

back to the solid bed of plant. The process runs
repeatedly until the extraction is completed.
Maceration Grind the plant materials into small particle to
increase the surface area for proper mixing with

The appropriate solvent is added in a closed vessel

and the plants are soaked for several days at room
temperature till the cellular structure is soften and
penetrated by the solvent and the soluble
constituents are dissolved and extracted out

The mixture then is filtered and the solid material

is pressed to recover large amount of extract

Occasional shaking in maceration facilitate

extraction by two ways;
(a) increase diffusion,
(b) remove concentrated solution from the sample
The plant materials are loaded in a flask

Water is added in sufficient

amount and then brought to boil. Hot
water and steam act as the main
influential factors to free bioactive
compounds of plant tissue.

Indirect cooling by water condenses the

vapor mixture of water and extract.

Condensed mixture flows from

condenser to a collector vessel.

Hydrodistillation involves three main

physicochemical processes;
Hydrodiffusion, hydrolysis and
decomposition by heat.
Extraction efficiency of any conventional method
mainly depends on the choice of solvents:

Polarity of
Ease of
the targeted
evaporation at
compound Choice of low heat

Low toxicity
/environmental Preservative
safety actions
Molecular affinity
between solvent and
solute, mass transfer,
Non-conventional Description/Principle
Extraction method assisted by ultrasound wave (20 kHz-
100 MHz).It passes through a medium by creating
compression and expansion. This process produces a
phenomenon called cavitation, which means production,
growth and collapse of bubbles.

Ultrasound assisted The implosion of cavitation bubbles can hit the surface of
Extraction the solid matrix and disintegrate the cells causing the
(UAE) release of the desired compound
Non-conventional Description/Principle
Extraction method assisted by Microwaves which are
electromagnetic fields (300 MHz to 300 GHz.)
Electromagnetic energy is converted to heat, thus, heating
the moisture inside the cells and evaporates, producing a
high pressure on the cell wall.

The pressure builds up inside the biomaterial which

modifies the physical properties of the biological tissues
Microwave assisted improving the porosity of the biological matrix. This would
Extraction allow better penetration of extracting solvent through the
(MAE) matrix and improved yield of the desired compounds
Non-conventional Description/Principle
Extraction method that used supercritical fluid as the
solvent. Supercritical state is a distinctive state and can
only be attained if a substance is subjected to temperature
and pressure beyond its critical point.

Carbon dioxide is considered as an ideal solvent for SFE.

Supercritical fluid Supercritical fluid possesses gas-like properties of
extraction diffusion, viscosity, and surface tension, and liquid-like
(SFE) density and solvation power. These properties make it
suitable for extracting compounds in a short time with
higher yields
Non-conventional Description/Principle
Extraction by the application of high pressure to remain
solvent liquid beyond their normal boiling point. High
pressure facilitates the extraction process.

PLE using organic liquid solvents at high temperature and

pressure to ensure the rapid extraction rate of compounds.
Pressurized liquid As the temperature increases the dielectric constant of the
extraction solvent decreases, consequently lowering the polarity of
(PLE) the solvent .

Thus, temperature could be used to match the polarity of

a solvent to that of the compounds of interest to be
recovered. The high pressure helps the extraction cells to
be filled faster and forces liquid into the solid matrix.
Non-conventional Description/Principle
Enzymes such as cellulase, β-glucosidase, xylanase,
β-gluconase, and pectinase help to degrade cell wall
structure and depolymerize plant cell wall poly saccha-
rides, facilitating the release of linked compounds .

The addition of specific enzymes during extraction

Enzyme-assisted enhances recovery by breaking the cell wall and
extraction hydrolyzing the structural polysaccharides and lipid bodies