Extraction of phytochemicals

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CONTENTS
•Definition
•Basics of Extraction
•Parameters for Selecting an Appropriate Extraction Method
•Steps Involved in the Extraction of Medicinal Plants
•The basic parameters influencing the quality of an extract
•Plant material
•Solvents used
•Choice of solvents
•Extraction procedures
•General methods of extraction.
•Dien Starck assembly for moisture content determination
•Microwave assitsed Extraction
•Countercurrent extraction
•Supercritical fluid Extraction

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Extraction (as the term is pharmaceutically used) is
the separation of medicinally active portions of plant
(and animal) tissues using selective solvents through
standard procedures.
DEFINITION
Extracts are the product
relatively complex mixture
of metabolites, in liquid or
semisolid state or (after
solvent recovery/removal)
in dry powder form.

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During extraction, solvents diffuse into the solid plant material
and solubilize compounds with similar polarity.
• Non polar solvents will dissolve non-polar compounds
• While polar solvents dissolve polar compound
• Solvent used is Menstrum
• Residue is Marc.
Extract Contains complex mixture
of many medicinal plant metabolites
Alkaloids,
Glycosides,
Terpenoids,
Flavonoids
Steroid
Triterpenoid
BASICS OF EXTRATION

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• Authentication.
• Foreign matter should be completely eliminated.
• Use the right plant part and, for quality control
purposes.
• Conditions used for drying the plant material
largely depend on the nature of its chemical
constituents.
• Powdered plant material should be passed
through suitable sieves to get the required
particles of uniform size.
Parameters for Selecting an Appropriate Extraction
Method

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The basic parameters influencing the quality of an extract
The basic parameters influencing the quality of an extract
Plant part used as starting material.
Solvent used for extraction
Extraction procedure
Variations in extraction methods affect quantity and secondary
metabolite composition of an extract depends upon
Type of extraction
Time of extraction
Temperature
Nature of solvent
Solvent concentration
Polarity

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In order to extract medicinal ingredients from plant material,
the following sequential steps are involved:
• Size reduction
• Extraction
• Filtration
• Concentration
• Drying
Steps Involved in the Extraction of Medicinal Plants

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•Plant based natural constituents part of the plant.
•Scientific analysis of components follows a logical
pathway.
•Plants are collected or supplied by local healers in
geographical areas where the plants are found.
•Fresh or dried plant materials used as a source for the
extraction.
•Plants are usually air dried to a constant weight
before extraction.
•Dry the plants in the oven at about 40°C for 72 h.
PLANT MATERIAL

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Properties of a good solvent in plant extractions
• Low toxicity
• Ease of evaporation at low temp.
• Promotion of rapid physiologic absorption of
the extract
• Preservative action
• Inability to cause the extract to complex or
dissociate.
CHOICE OF SOLVENTS

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Factors affecting the choice of solvent :-
•Quantity of phytochemicals to be extracted
•Rate of extraction
•Diversity of different compounds extracted.
•Diversity of inhibitory compounds extracted.
• Ease of subsequent handling of the extracts.
•Toxicity of the solvent in the bioassay process.
•Potential health hazard of the extracts.
•The choice will also depend on the targeted
compounds to be extracted.

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GENERAL METHODS OF HERBAL
PLANT EXTRACTION
 Maceration,
 Infusion,
 Percolation,
 Digestion,
 Decoction,
 Hot continuous extraction
(Soxhlet),
 Counter-current extraction,
 Microwave-assisted extraction,
 Ultrasound extraction
(sonication),
 Supercritical fluid extraction,
 Phytonic extraction (with
hydrofluorocarbon solvents).
 Hydrodistillation techniques
(water distillation, steam
distillation, water and steam
distillation),
 Hydrolytic maceration followed
by distillation, expression and
enfleurage (cold fat extraction)
 Headspace trapping,
 Solid phase micro-extraction,
 Protoplast extraction,
 Microdistillation,
 Thermomicrodistillation,
 Molecular distillation.
Extraction techniques For
aromatic plants

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Types of Extraction
Successive Fractionation
Same drug material
Extracted using different
Solvents
Solvents chosen according to
increasing polarity
Crude extract prepared with
single solvent
Fractionated with different
solvents
drug material

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Infusion
• Fresh infusions are prepared by macerating
the crude drug for a short period of time with
cold or boiling water.
• These are dilute solutions of the readily soluble
constituents of crude drugs

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1. The drug is usually coarsely powdered, very fine powder
being avoided (50 gm).
2. Moisten the drug in a suitable vessel, provided with a cover,
with 50 ml of cold water.
3. Allow to stand for 15 minutes.
4. Then add 900 ml of boiling water, cover the vessel tightly.
5. Allow it to stand for 30 minutes.
6. Then strain the mixture, pass enough water to make the
infusion measure 1000 ml.
7. Some drugs are supplied in accurately weighed in muslin
bags for preparing specific amounts of infusion.
8. If the activity of the infusion is affected by the temperature of
boiling water, cold water should be used.
9. As the infusions are not stable, they should be made freshly
and used.
Infusion Process

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Decoction
•Method is used for the extraction of the water soluble and
heat stable constituents
•Crude drug is boiled in water for 15 minutes,then cooling,
straining and passing sufficient cold water through the drug
to produce the required volume.

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Digestion
• This is a kind of maceration in
which gentle heat is applied during
the maceration extraction process.
• It is used when moderately elevated
temperature is not objectionable
and the solvent efficiency of the
menstrum is increased.
• Heat Reflux-

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In maceration (for fluid extract), whole or coarsely
powdered plant-drug is kept in contact with the
solvent in a stoppered container for a defined
period with frequent agitation until soluble matter is
dissolved.
Maceration.

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Plant Material (Crushed or cut small or Moderately coarse powder)
Placed in a bottle 1 liter )
Whole of the selected solvent (closed vessels maceration
menstrum)added
Allowed to stand for seven days shaking occasionally
Liquid strained off Solid residue (mark) pressed
(Recover as much as occluded solution)
Strained and expressed liquids mixed
Clarified by subsidence or filtration

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Types of Maceration
1. Simple maceration of organized drugs
2. Simple maceration of Unorganized drugs
3. Multiple maceration (Single, Double
Triple)

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Processes for Organized Drugs
(e.g. Barks, Roots)
Processes for Unorganized Drugs
(e.g. Gum- resin)
(i) Drug + whole of menstruum (i) Drug + 4/5 ths (in most cases) of
menstruum
(ii) Shake occasionally during 7 days (ii) Shake occasionally during 2 to 7
days as specified
(iii) Strain of liquid, and press the
marc
(iii) Decant the liquid. Marc is not
pressed
(iv) Mix the liquid, clarify by subsidence
for filtration
Filtrate is not adjusted to volume
(iv) Filter the liquid and pass more
menstrum through
filter to volume
Preparations made by this process
Vinegar of squill, B.P.C
Oxymel of Squill, B.P.C
Tincture of Orange I.P
Tincture of Capsicum, B.P.C
Compound Tincture of Gentian
Tincture of Lemon
Tincture of Squill, B.P.C
Preparations made by this process
Compound Tincture of Benzoin
Tincture of Myrrh, B.P.C
Tincture of Tolu, B.P.C
Maceration Processes for Organized and Unorganized Drugs

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Percolation
This is the procedure used most frequently to extract active
ingredients in the preparation of tinctures and fluid extracts

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Percolation Process divided in following steps
•Size reducation or
communiation of the
drug
•Imbibation
•Packing
•Maceration
•Percoation

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Organized vegetable drug is taken in powdered form.
Uniform moistening of the powdered vegetable drugs with menstruum
for a period of 4hours in a separable vessel (Imbibition).
Packed evenly into the percolator.
A piece of filter paper is placed on surface followed by a layer of clean
sand so that top layers of drugs are not disturbed.
Sufficient menstruum is poured over the drug slowly and evenly to
saturate it, keeping the tap at bottom open for passing of occluded gas
to pass out.
Percolation Process

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Sufficient menstruum is also added to maintain a small layer above the
drug and allowed to stand for 24 hours.
After maceration, the outlet is opened and solvent is percolated at a control
rate with continuous addition of fresh volume.
75% of the volume of the finished product is collected.
Marc is pressed and expressed liquid is added to the percolate giving 80%
to 90% of the final volume.
Volume is adjusted with calculated quantities of fresh menstruum.
Evaporation and concentration to get finished products by applying suitable
techniques and apparatus

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Continuous Hot Percolation extraction(Soxhlet)
1. Stirrer
2. Still pot
3. Distillation
path
4. Thimble
5. Solid
6. Siphon top
7. Siphon exit
8. Expansion
adapter
9. Condensor
10.Cooling
water in
11.Cooling
water out

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Ultrasound-assisted Soxhlet extraction:
Expeditive approach for solid sample treatment
Application to the extraction of total fat from oleaginous seeds

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•
1: Stirrer bar/anti-bumping
granules
2:RBF (Still pot)
3: Fractionating column
4: Thermometer/Boiling
point temperature
5: Condenser
6: Cooling water in
7: Cooling water out
8: Graduated (Burette) tube
9: Tap
10: Collection vessel
Dean-Stark Assembly-For
Moisture content determination

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• This equipment is usually used in azeotropic distillations.
• A common example is the removal of water generated
during a reaction in boiling toluene.
• An azeotropic mixture of toluene and water distills out of
the reaction
• Only the toluene (density=0.865 g/ml) returns, since it
floats on top of the water (density=0.998 g/cm3), which
collects in the trap.
• Hence Dean-Stark method is commonly used to measure
moisture content
Two types of Dean-Stark traps exist
1)For solvents with a density less than water
2)For solvents with a density greater than water

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1. Thoroughly clean the receiving tube and the condenser
of the apparatus, rinse with water and dry.
2. Introduce 200 ml of toluene R and about 2 ml of water
into a dry flask.
3. Heat the flask to distil the liquid over a period of 2 hours
4. Allow to cool for about 30 minutes and read off the
volume of water to an accuracy of 0.05 ml (first
distillation).
5. Weigh accurately a quantity of the material expected to
give about 2-3 ml of water and transfer to the flask. (For
weighing material with a paste-like character, use a boat
of metal foil.)
6. Add a few pieces of porous porcelain and heat the flask
gently for 15 minutes.
Steps Invovled in determination of moisture by
Dien Starck assembly

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7. When boiling begins, distil at a rate of 2 drops per
second until most of the water has distilled over, then
increase the rate of distillation to about 4 drops per
second.
8. As soon as the water has been completely distilled,
rinse the inside of the condenser tube with toluene R.
9. Continue the distillation for 5 more minutes, remove the
heat, allow the receiving tube to cool to room
temperature and dislodge any droplets of water
adhering to the walls of the receiving tube by tapping
the tube.
10.Allow the water and toluene layers to separate and read
off the volume of water (second distillation).
11.Calculate the content of water as a percentage using
the formula:
100(n1 − n)
w
Where
w = the weight in g of the material being examined
n = the number of ml of water obtained in the first distillation
n1 = the total number of ml of water obtained in both distillations

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Extraction of Volatile Oil

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Presence of Essential Oil in Plant parts.

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Methods of extraction of oils from plant parts

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•A) Direct steam distillation:
Cut the drug freshly
Take it into distillation flask
Pass generated steam through
material
Then pass volatile le oil content
through water condenser
Volatile oil get collected in flask
depending on whether oil is
lighter/heavier than water.
EXTRACTION OF VOLATILE OILS

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A.Water distillation :
Applicable to those material whose constituent are not degreased by
boiling up to 100 degree. Eg. Turpentine oil.
B. Water & steam distillation:
Applicable to those plant material whose constituent undergoes
degradation by direct boiling e.g.. Clove oil, cinnamon oil.
C. Direct steam distillation:
Applicable to fresh drug that is loaded with sufficient natural
moisture hence no maceration is required. e.g.. Peppermint oil .
B) Expression:
This method is used to obtain volatile oils which are present
in rind of fruit by extrusion application of pressure to preserve
natural fragrance otherwise they get deteriorated by distillation
process.

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Sponge method:
Wash citrous fruit to remove dirt
Cut into halves to remove juice completely
Rind is turned inside out by hand
When secretory glands ruptures rind is squezed
Finally collect oozed volatile oil with the help of sponge &
squeeze it in a vessel.

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Scarification process(ecuelle a piquer):it is specialy
designed apparatus.
Keep freshly washed lemons into apparatus
Rotate it repetedly
When oil glands are punctured oil get discharged
Collect discharged oil transfer it to another vessel& keep it
Decant clear oil
Finally filter it get clear volatile oil

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Rasping process:-
Remove outer surface of peel of citrus fruit containing oil gland.
Put it in horsehair bags
Press it strongly
Oil stred in oil glands get ooze out which has turbid apperence
Allow it to stand
Finally oil seprated put which may be decanted
Filter it subsequently
Mechanical process:
This method is based on above principle. in this method heavy duty centrifuge
devices are used so that there is easy separation of oil/water emulsion takes place.

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• Extraction Method
Extraction process is used for such plant sources which either
content very small amount of volatile oils or the oil contents are
extremely susceptible to decomposition by the exposure to steam.
In such cases recovery of volatile oil is not commercially feasible.
In general extraction of volatile oils from natural sources is carried
out by two different methods.
A. Extraction with
volatile solvent:
e.g. Hexane, benzene.
B. Extraction with
non volatile solvents:
e.g. tallow, olive oil.

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• A. Extraction with volatile solvents:-
 Plant material extracted with low boiling volatile solvents. Like
benzene, hexane , pet. Ether. By adopting the method of hot
continuous extraction. (Soxhlet extraction)
 Resulting volatile oil removed under reduced pressure.
• B.Extraction with non volatile solvents:
 This process is employed for the preparation of finest brands of
perfume oil, i.e. natural flower oil.
 There are three methods used for the extraction of volatile oils
from flowers with non vol. solvents.

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Enfleurage:
 A glass plate is covered with a thin coating of especially prepared and odourless
fat (called a chassis).
 The freshly cut flowers are individually laid on to the fat which in time becomes
saturated with their essential oils.
 The flowers are renewed with fresh material.
 Eventually the fragrance-saturated fat, known as pomade, may be treated with
alcohol to extract the oil from the fat.

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Pneumatic method :
The basic principle of this method is very much closer to enflurage
method
Current of warm air is passed through flowers.
It is loaded with suspended volatile oil particles.
Then pass it through a fine spray of melted fat in which volatile oil
get absorbed.
Maceration:
 Fresh flowers are gently & carefully heated in melted fat.
 Stir it frequently until complete exhaustion takes place
 Flowers are then stained & squeezed exuded
 Fat is returned to main bulk of fat unless &until a desired conc is
achieved .
 Cool volatile oil containing fat recover it by three successive
extractions .

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Introduction to Newer Techniques
of Extraction

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1. In counter-current extraction (CCE), wet DRUG material is
pulverized and produce a fine slurry.
2. Here the material to be extracted is moved in one direction
within a cylindrical extractor where it comes in contact with
extraction solvent.
3. The further the starting material moves, the more concentrated
the extract becomes.
4. Complete extraction is thus possible when the quantities of
solvent and material and their flow rates are optimized.
5. Finally, sufficiently concentrated extract comes out at one end of
the extractor while the marc falls out from the other end.
Counter-current Extraction (CCE)

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Solvent Inlet Drug Slurry Inlet
Residue out
Extract out
Counter-current Extraction (CCE)

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Advantages of Counter-current Extraction
•Unit quantity of plant material can be extracted with
much smaller volume of solvent.
•CCE is commonly done at room temperature this
method spares thermo labile compounds from
exposure to heat
•As pulverization of drug is done under wet condition
heat generated during comminution is neutralized.
•This method is more efficient than hot percolation.

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Appliction of Counter-current Extraction
1. Oil is to be extracted from soya beans in a counter
current stage-contact extraction apparatus, using
hexane.
2. application have been concerned with the purification
and separaion of organic or biochemical compound.
3. application in inorganic chemistry seem to confined to
separation of the radio-nuclides and a new techniques
for the determination of trace elements in geological
material.

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 Process of separating one component from another using
supercritical fluids as the extracting solvent.
 Extraction is usually from a solid drug, but can also be from
liquids.
 SFE can be used as a sample preparation step for analytical
purposes
 For larger scale to either strip unwanted material from a
product or collect a desired product.
 Carbon dioxide (CO2) is the most used as supercritical
fluid, sometimes modified by co-solvents such as ethanol or
methanol.
 Extraction conditions for Supercritical carbon dioxide
are above the critical temperature of 31°C and critical
pressure of 74 bar.
Supercritical Fluid Extraction (SFE)

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Introduction of feed into extractor (solid feed) or extractor in
modified column either co-currently or counter-currently
Formation of mobile phase: mixing of solutes with supercritical
fluid.
Exposure of mobile phase to pressures (50-500 atm) and
temperatures (ambient to 300°C) near or above the critical
point for enhancing the mobile phase solvating power.
Isolation of dissolved solute by precipitation
Eg. CO2 in vapour form is compressed into a liquid before
becoming supercritical and then extraction takes place.
Supercritical Fluid Extraction (SFE) Process

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COMPONENTS OF SFE
1. Fluid reservoir (gas cylinder in case of CO2)
2. Pump – Reciprocating pump – Syringe pump (pulse-free flow at
large range of flow rates)
3. Extraction cell/column (stationary phase) – Usually stainless steel
Chamber or vessel in compartment – Capable of withstanding high
pressure (300-600 atm) [for solids] – Open tubular capillary columns or
packed columns [liquids]
4. Restrictor – Maintaining pressure change inside the extraction vessel
Two types- – fixed (linear restrictor, tapered desire, integral restrictor,
ceramic frit restrictor, metal restrictor) – Variable (variable nozzle, back
pressure regulator)
5. Collector (trapping system)
6. Detectors (flame ionization detector of gas chromatography

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Supercritical fluid Extractor (SFE)

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Advantages and Disadvantages of (SFE)
Advantages Disadvantages
High diffusion rate than liquid
solvents
CO2 has low polarity and hence
cant extract polar compounds
Lower viscosities than liquid
solvent
Presence of water / moisture may
cause problems
High efficiency compared to
general methods
Unpredictability of matrix effect
Suitable for Thermolabile
compounds
Need of specilized /expensive
equipment
Low polarity of CO2 can be
modified by co-solvent
Sterile and Bateriostatic
Non combustible and non-
explosive environment friendly

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• Prolonged time (penetration of SCF into the interior of a
solid is rapid, but solute diffusion from the solid into the
SCF).
• Modeling is inaccurate
• Scale is not possible (due to absence of fundamental,
molecular-based model of solutes in SCF)
• Expensive
• Consistency & reproducibility may vary in continuous
production
LIMITATIONS

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• Adequate contact time (for penetration of solvent into
solid particles and diffusion of solute from inside the solid
particles to solvent)
• Equilibrium should be achieved (i.e. proper flow of
solvent such that concentration of dissolved solutes in the
solvent phase will be below the solubility of solute in
solvent)
PRECAUTIONS

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Food science
• Fat and oil samples in meat, egg, meals, chocolate, dairy
products, seeds and food snacks,
Natural products
• Flavors & spices of ginger, eucalyptus, soyabean, coffee,
soybean, basil, lime peels, potato chips, popcorn
By-products recovery
• Fruit and vegetable waste
Application of Supercritical Fluid Extraction (SFE)

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Application of Supercritical Fluid Extraction (SFE)

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MICROWAVE THEORY
 Microwaves are non-ionizing electromagnetic waves
 They have frequency between 300MHz to 300 GHz
 Microwaves are positioned between the X-ray and infrared
rays in the electromagnetic spectrum.
 Microwaves serves two major purposes:
– Communication – Energy vectors
Microwave-assisted extraction
The energy vector application is the direct action of waves on
materials that has the ability to convert a part of the absorbed
electromagnetic energy to heat energy.
Microwaves are made up of two oscillating perpendicular
field’s i.e. – Electric field – Magnetic field

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Microwave-assisted extraction

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In MAE, heating occurs in a targeted and selective manner
with practically no heat being lost to the environment as the
heating occurs in a closed system.
This unique heating mechanism significantly reduces the
extraction time (usually less than 30min) as compared to
Soxhlet.
EXTRACTION PRINCIPLE
• Dried plants contain a minute microscopic trace of moisture
which serves as target for microwave heating.
• High temperature and pressure is generated inside the
oven.
• High temperature causes dehydration of cellulose which
accounts for its reduction of mechanical strength.

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Principle of the method and heating mechanis

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MAE process
Microwave radiation enters plant cell
Moisture get heated up
Moisture evaporates
Generation of tremendous pressure on cell wall
Swelling of plant cell
Rupture of the cell
Leaching out of phyto-constituents
This phenomenon can be intensified if the plant matrix is
impregnated with solvents with higher heating efficiency
under microwave.

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Accelerated solvent extraction
Soxhlet extraction

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CLOSED VESSEL
• With closed vessel extraction,
pressurized microwave assisted
extraction is performed under
pressure (with or without
regulation).
• The pressure allows
temperatures above boiling
points of the solvents to be
reached enhancing speed and
efficiency.
• To avoid overpressure, power,
temperature and pressure can be
controlled.
• When dealing with thermo-labile
compounds, high temperature
causes degradation of analytes.
Therefore, the open system is
chosen.

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Disadvantages of
closed-vessel system
1.High pressure used poses
safety risks
2.Usual constituent material
of the vessel does not allow
high solution temperatures
3.Addition of reagents is
impossible since it is a single
step procedure
4.Vessel must be cooled
down before it can be opened
to prevent loss of volatile
constituents
Advantages of closed-
vessel system
1.Decreased in extraction time
2.Loss of volatile substances is
avoided
3.Less solvent is required
because no evaporation occurs
4.No hazardous fumes during
acid microwave since it is a
closed vessel

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OPEN VESSEL
• Extraction is made at
atmospheric pressure.
• The maximum possible
temperature is determined
by the boiling point of the
solvent at that pressure.
• Loss of vapor's is
prevented by the presence
of a cooling system on the
top of the extraction vessel
that causes condensation
of solvent vapors.
• Heating is homogenous
and efficient.

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Disadvantages of
closed-vessel system
1.This method is less precise
than in close-vessel system
2.The sample throughput is
lower as open system cannot
process many samples
simultaneously
3.Require longer time to achieve
same results as for closed
system
Advantages of closed-
vessel system
1.Increased safety
2.Addition of reagent is possible
3.Vessels made of various
material can be used
4.Excess solvent can be
removed easily
5.Ability to process large
samples
6.No requirement for cooling
down or depressurization
7.Low cost of equipment
8.Suitable for thermolabile
products

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FACTORS AFFECTING MAE
• Extraction time
• Microwave power
• Matrix characteristics
• Temperature

Extraction of phytochemicals

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