METHODS OF ISOLATION oF MAJOR PROACTIVE CONSTITUENTS FROM
MEDICINAL PLANTS
(Cold Extraction and Soxhlet Extraction)
Dr. Shah Rafiq
Medicinal plants contain many kinds of chemical compounds. Some are primary metabolites
(sugars, proteins, lipids) and others are secondary metabolites, which are responsible for
pharmacological activity.
Major bioactive constituents
• Alkaloids (e.g., morphine, atropine, quinine, reserpine)
•
Glycosides (e.g., digoxin, sennosides, aloin)
•
Flavonoids and phenolics (e.g., quercetin, rutin, tannins)
•
Terpenoids and essential oils (e.g., menthol, eucalyptol)
•
Saponins, steroids, resins and coumarins
Extraction is the process of separating the medicinally active constituents of plant tissue from
the inactive or inert material (cell wall matter, cellulose and so on) using a suitable solvent and
a suitable method. The product is called the extract, and the leftover plant material is the
marc. The solvent used is also called the menstruum.
Objectives of extraction
1. To obtain therapeutically active components in concentrated form.
2. To remove inert or unwanted material.
3. To prepare the crude drug for standardisation and formulation (tinctures, fluid
extracts, dry extracts).
4. To prepare the sample for isolation and structure elucidation of pure compounds.
General Scheme for Isolation of Bioactive Compounds
1. Collection and authentication of plant material
2. Drying and size reduction
3. Selection of solvent
4. Extraction (cold or hot method)
5. Filtration and concentration of the extract
6. Fractionation (liquid-liquid partitioning, precipitation)
7. Purification (column chromatography, preparative TLC, HPLC, crystallisation)
8. Characterisation (TLC, UV, IR, NMR, MS)
Pre-extraction Preparation of Plant Material
a) Collection and authentication
• The correct species, plant part, season and stage of growth must be used, because the
content of constituents varies with these factors.
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Dr. Shah Rafiq
•
A botanist should authenticate the plant, and a voucher specimen should be kept.
b) Drying
• Drying stops enzymatic degradation and microbial growth.
•
Shade drying is used for thermolabile compounds and volatile oils.
•
Oven drying at 40 to 50 °C or sun drying may be used for stable constituents.
•
Fresh material is sometimes used directly for volatile constituents, but its water
content dilutes the solvent.
c) Size reduction (grinding or powdering)
• Grinding increases the surface area and ruptures cell walls, which improves solvent
penetration.
•
The powder should be moderately coarse to fine (commonly No. 20 to No. 60
sieve).
•
Very fine powder can clog percolators and thimbles and make filtration difficult.
Coarse powder gives poor extraction efficiency.
d) Defatting (when required)
• Seeds and other oily materials are first extracted with a non-polar solvent such as
petroleum ether or hexane, so that fats do not interfere with later extraction.
Selection of Solvent
The governing rule is "like dissolves like": polar compounds dissolve in polar solvents and
non-polar compounds in non-polar solvents.
Characteristics of an ideal solvent
• Selective for the target compounds
•
Chemically inert (does not react with the extract)
•
Low boiling point, so it is easily removed
•
Non-toxic, non-flammable and inexpensive
•
Does not cause complex formation or degradation
Solvents in increasing order of polarity
Solvent
Polarity
Typically extracts
Petroleum ether / Hexane
Very low
Fats, waxes, oils, sterols, carotenoids
Chloroform
Low
Alkaloid bases, terpenoids, some glycosides
Diethyl ether
Low
Volatile oils, alkaloids, terpenoids
Ethyl acetate
Medium
Flavonoid aglycones, phenolics
Acetone
Medium-high
Flavonoids, phenolics, tannins
Ethanol
High
Alkaloids, glycosides, flavonoids, tannins,
saponins
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Solvent
Polarity
Typically extracts
Methanol
High
Broad range, including polar glycosides
Water
Very high
Sugars, glycosides, tannins, saponins, gums,
mucilage
Note: Ethanol and water-ethanol mixtures are the most widely used in industry because they
are relatively safe. Methanol extracts a wide range of compounds but is toxic.
Classification of Extraction Methods
A. Cold (room temperature) methods: maceration (simple, multiple and with agitation) and
percolation.
B. Hot methods: decoction, infusion and digestion, Soxhlet (continuous hot) extraction,
reflux extraction, steam distillation.
C. Modern methods: ultrasound-assisted extraction (UAE), microwave-assisted extraction
(MAE), supercritical fluid extraction (SFE, with CO2), pressurized liquid extraction.
This lecture covers cold extraction and Soxhlet extraction.
Cold Extraction
Cold extraction is carried out at room temperature without applying heat. It is best suited
to thermolabile constituents and is therefore called a "gentle" method.
Maceration
Principle: The coarse or powdered drug is kept in contact with the solvent (menstruum) in a
closed vessel for a set period,
with occasional shaking or
stirring. The solvent penetrates
the cell wall and dissolves the
soluble constituents, and the
solution diffuses out until
equilibrium
is
reached
between the concentration
inside and outside the cells.
Apparatus: A wide-mouthed,
closed container (glass jar or
stoppered bottle) made of inert
material.
Procedure (simple maceration)
1. Place the weighed, coarsely powdered plant material in a closed container.
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2. Add the solvent (commonly about 3 to 5 times the weight of the drug, or enough to
cover it completely).
3. Close the vessel and keep it at room temperature for 3 to 7 days with frequent shaking
or stirring.
4. Strain or filter the liquid. Press the marc to recover the remaining liquid.
5. Combine the liquids, clarify by filtration or by standing and decanting, and
concentrate.
Types of maceration
• Simple maceration: The procedure above, used for most drugs and for tinctures.
•
Multiple maceration (double or triple): The solvent is divided into portions. The
drug is macerated with the first portion and strained, and the marc is macerated again
with the second portion. This gives more complete extraction than one large volume.
•
Maceration with agitation (dynamic maceration): Occasional or mechanical
stirring, for example with a shaker, increases the rate of diffusion and shortens the
time.
Advantages
• Simple, inexpensive and needs minimal equipment
•
Suitable for thermolabile constituents
•
Little chance of thermal decomposition
Disadvantages
• Time-consuming (days)
•
Extraction is often incomplete, because equilibrium is reached and some drug is left
in the marc
•
Uses a large volume of solvent
•
Solvent may be lost by evaporation if the container is not tightly closed
Examples of use: Tincture of ginger, tincture of orange, tincture of tolu, and extraction of
colored pigments and thermolabile flavonoids.
Percolation
Principle: A continuous downward flow of fresh solvent through a bed of drug in a conical
vessel (percolator). Because fresh solvent is always in contact with the drug, the concentration
gradient stays high, giving more complete extraction than maceration.
Apparatus: A percolator, a narrow conical or cylindrical vessel with a stopcock at the bottom.
Procedure
1. Moistening (imbibition): The powder is moistened with enough solvent to make it
evenly damp and left for about 4 hours in a closed container so that it swells and the
solvent penetrates.
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2. Packing: The moistened drug is packed uniformly into the percolator over a cotton or
filter pad, neither too loose nor too tight.
3. Maceration (soaking): Solvent is added until it stands above the drug bed. The
stopcock is closed and the drug is macerated for about 24 hours.
4. Percolation: The stopcock is opened and the extract is drawn off slowly, usually at a
rate of about 1 mL per minute per 1000 g of drug. Fresh solvent is added continuously
so that the drug is always covered by a layer of solvent.
5. Collection continues until the drug is exhausted, meaning the percolate no longer
gives a test for the constituents.
Reserved percolation: The first portion of the strong percolate is set aside as "reserved". The
later, weaker percolate is concentrated at low temperature and added to the reserved portion.
This avoids heating the most concentrated extract.
Advantages
• More complete extraction than maceration
•
Suitable for potent drugs and for preparing fluid extracts and tinctures
Disadvantages
• Needs more skill and more solvent
•
The extract may be dilute
•
Not suitable for very fine powders (channeling or blocking of the bed)
Soxhlet (Continuous Hot) Extraction
Principle
Soxhlet extraction is a continuous extraction with recycled
solvent. The solvent is heated, its vapour condenses, and the
condensed liquid repeatedly percolates through the solid sample
held in a thimble. When the thimble chamber is full, a siphon
empties it back into the flask. Each cycle uses fresh, distilled
solvent, so extraction proceeds as though a large volume of solvent
were used, while only a small volume is actually needed. The
extracted compounds are non-volatile, so they accumulate in the
flask as pure solvent is redistilled.
Apparatus
A Soxhlet apparatus has three main parts:
1. Round-bottom flask at the bottom, containing the solvent
and boiling chips or a stir bar, sitting on a heating mantle
or water bath.
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2. Soxhlet extractor (extraction chamber) in the middle, containing the thimble with
the sample, a vapour (bypass) side arm and a siphon tube.
3. Condenser (reflux condenser, usually a water-cooled Liebig or Allihn type) on top.
The thimble is made of thick filter paper or cellulose. It must be porous enough for the solvent
to pass but must retain the plant powder.
Procedure (Step by Step)
1. Weigh the dried, coarsely powdered plant material (for example 20 to 50 g).
2. Place the powder in the thimble and plug the top with cotton wool so that no powder
floats out.
3. Place the thimble in the extraction chamber, ensuring the drug is below the top of the
siphon arm.
4. Fill the round-bottom flask to about one-half to two-thirds with solvent and add
boiling chips.
5. Assemble the apparatus and start the flow of cooling water through the condenser.
6. Heat the flask at a controlled rate. The solvent boils, its vapor rises through the side
arm to the condenser, condenses, and drips onto the drug in the thimble.
7. The chamber fills with warm solvent, which dissolves the compounds. When the level
reaches the top of the siphon, the whole volume is siphoned back into the flask. This
completes one cycle.
8. Repeat for a fixed number of cycles or hours (commonly 6 to 24 hours, at about 4 to
6 siphonings per hour) until the drug is exhausted. The solvent in the extraction
chamber then becomes colorless and gives a negative test for the constituents.
9. Stop heating, allow to cool and dismantle the apparatus.
10. Concentrate the extract from the flask, usually by rotary evaporator under reduced
pressure, then dry it in a desiccator or vacuum oven.
Factors Affecting Soxhlet Extraction
• Particle size: smaller particles give better extraction, but very fine powder can block
the thimble
•
Solvent type and polarity: must match the target compounds
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Dr. Shah Rafiq
•
Extraction time and number of cycles
•
Temperature: governed by the boiling point of the solvent
•
Sample-to-solvent ratio
•
Moisture content of the sample: high moisture reduces efficiency with non-polar
solvents
•
Rate of heating: too fast a rate causes flooding and poor contact
Advantages
• The solvent is recycled, so a small volume gives an efficient, exhaustive extraction
•
Very high extraction efficiency, more complete than maceration
•
The drug is always in contact with fresh, warm solvent
•
Requires little supervision once set up
•
Very suitable for non-polar and moderately polar compounds such as lipids,
essential oils and alkaloids
•
Simple and widely used as a standard reference method
Disadvantages
• Thermal degradation of heat-sensitive compounds, because the extract stays at the
boiling point of the solvent for a long time
•
Long extraction times (hours)
•
Large amounts of solvent are consumed overall, and evaporating them takes energy
•
Not suitable for thermolabile constituents (some glycosides, volatile oils and certain
flavonoids)
•
The recycled solvent gives no agitation, so it is less suitable for compounds that are
poorly soluble in the solvent
•
Unsuitable for solvents with high boiling points, or for solvent mixtures whose
composition changes on distillation
•
Difficult to scale up for large industrial batches
•
Flammable solvents pose a fire hazard, so heating mantles or water baths should be
used rather than open flames
Common Uses
• Determination of fat or oil content (petroleum ether)
•
Extraction of alkaloids (for example from Cinchona, Datura, Nux vomica)
•
Defatting of seeds before further extraction
•
Extraction of steroids, terpenoids and lipophilic phytoconstituents
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Comparison of Cold Extraction and Soxhlet Extraction
Feature
Cold extraction (maceration /
percolation)
Soxhlet extraction
Temperature
Room temperature
Boiling point of the solvent
Solvent handling
Static, or single pass (percolation)
Recycled continuously
Time
Days (maceration); hours to a day
(percolation)
Hours
Solvent volume
Large
Small (recycled)
Efficiency
Lower (maceration) to moderate
(percolation)
High, near exhaustive
Suitability
Thermolabile compounds
Thermostable compounds
Risk of
decomposition
Low
Possible
Equipment
Simple and cheap
Glassware, heat source, water
supply
Scale
Easy to scale
Difficult to scale up
Post-extraction Steps
1. Filtration: Filter the extract through filter paper or muslin cloth to remove particles.
2. Concentration: Remove the solvent using a rotary vacuum evaporator at low
temperature and reduced pressure (about 40 to 50 °C), which protects heat-sensitive
compounds.
3. Drying: Dry in a desiccator, vacuum oven, or by lyophilization (freeze-drying) to
obtain a dry extract.
4. Fractionation: Separate the crude extract into groups of compounds by liquid-liquid
partition between immiscible solvents (hexane, chloroform, ethyl acetate, butanol,
water), acid-base treatment (for alkaloids), or precipitation with solvents or reagents.
5. Purification: Column chromatography (silica gel or alumina), preparative TLC,
HPLC and recrystallization.
6. Identification: TLC, UV, IR, NMR and mass spectrometry.
Example: Isolation of Alkaloids by Soxhlet Extraction
1. Powder the dried plant material and moisten it with alkali (ammonia or lime). This
liberates free alkaloid bases from their salts.
2. Extract with an organic solvent (chloroform or ether) in a Soxhlet apparatus.
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3. Shake the organic extract with dilute acid (dilute HCl or H2SO4). The alkaloids move
into the aqueous layer as water-soluble salts, and neutral impurities remain in the
organic layer.
4. Make the aqueous layer alkaline. The free alkaloid bases precipitate or can be
extracted back into an organic solvent.
5. Concentrate and purify by chromatography or crystallisation.
II. Preparation of Active Content-Enriched Extracts
Medicinal plants contain hundreds of chemical constituents, but usually only a small group of
them is responsible for the therapeutic effect. A crude herbal extract therefore carries a large
proportion of inert or unwanted material, such as chlorophyll, waxes, sugars, proteins, and
cellulose fragments, alongside the constituents of interest. An active content-enriched extract
is an extract that has been deliberately processed so that the percentage of the bioactive
constituent, or of a class of constituents, is markedly higher than in the starting plant material
or in a simple crude extract.
Key definitions
Term
Meaning
Crude extract
Concentrated preparation obtained by treating plant material with a
solvent and removing the solvent; contains the full spectrum of
extractable compounds.
Enriched extract
Extract in which the content of the active compound or class (for
example, total flavonoids, total saponins, curcuminoids) has been raised
by additional processing steps.
Standardized extract
Extract adjusted to a defined content of a marker or active constituent,
within stated limits, by blending or by process control. An enriched
extract is often also standardised.
Fraction
A portion of an extract separated on the basis of polarity, size, acidity or
affinity (for example the ethyl acetate fraction).
Isolated compound
A single, purified chemical entity (for example, berberine, quercetin);
purity is usually above 95%.
Active constituent
Compound(s) responsible for the therapeutic activity.
Marker
Chemically defined constituent used for quality control, whether or not
it is therapeutically active.
Drug-extract ratio
(DER)
Quantity of plant material used to make a unit quantity of extract; for
example, 10:1 means 10 kg of herb gives 1 kg of extract.
Why enrich extracts?
•
Consistent therapeutic dose: natural variability in plant content is reduced, so each
dose delivers a predictable amount of active.
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•
Smaller dose volume: a higher active concentration allows a capsule or tablet of
practical size instead of large amounts of powder.
•
Improved efficacy and bioavailability: removal of interfering matrix components
such as tannins or polysaccharides can improve absorption and pharmacological
response.
•
Improved safety: unwanted or toxic compounds (for example pyrrolizidine alkaloids,
aristolochic acid, excess furanocoumarins) can be depleted deliberately.
•
Better stability and formulation behaviour: less hygroscopic and less sticky
material; improved flow, compressibility and shelf life.
•
Regulatory and commercial requirements: nutraceutical and phytopharmaceutical
products commonly require a declared minimum marker content.
•
Economic advantage: lower transport, storage and processing costs per unit of
active.
General Workflow
Preparation of an enriched extract follows a logical sequence. Every stage influences the yield
and the final marker content, so errors at an early stage cannot be corrected later.
1. Selection and authentication of the plant material (botanical identity, correct part,
chemotype).
2. Collection, post-harvest handling and drying under conditions that protect the
actives.
3. Size reduction to an optimum particle size.
4. Pre-treatment where necessary (defatting, enzymatic inactivation, hydrolysis,
moistening).
5. Primary extraction with a suitable solvent and method.
6. Filtration, clarification and concentration of the miscella (liquid extract).
7. Enrichment or fractionation (partitioning, adsorption, precipitation,
chromatography, membranes and so on).
8. Final concentration and drying (vacuum drying, spray drying, freeze drying).
9. Standardisation with carriers or by blending batches to a fixed marker content.
10. Quality control, packaging and stability testing.
Raw Material Preparation
i.
Selection and authentication
The quality of an extract cannot exceed the quality of the raw material. The following
parameters must be confirmed before extraction begins:
•
Botanical identity: macroscopic and microscopic examination, comparison with a
herbarium specimen, and chemical fingerprinting by TLC or HPTLC.
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•
Plant part: the active content differs among root, rhizome, leaf, bark, flower, fruit and
seed. For example, withanolides are concentrated mainly in Withania somnifera roots
and leaves, whereas piperine is concentrated in the fruits of Piper nigrum.
•
Chemotype and geographic origin: the same species may occur as chemotypes with
different major constituents.
•
Age and season of harvest: alkaloids, glycosides and volatile oils change with the
developmental stage and the time of day. Many drugs are best collected at flowering or
at maximum leaf development.
•
Foreign matter, adulterants, moisture, ash values, heavy metals, pesticide
residues, aflatoxins and microbial load as prescribed by pharmacopoeial
monographs.
ii.
Drying
Drying stops enzymatic degradation, prevents microbial growth and allows storage. The
method must suit the constituent class.
Method
Conditions
Suitability
Shade drying
Ambient, good ventilation, no direct
sunlight
Aromatic leaves and flowers, volatile oil drugs
Sun drying
Direct sun, several days
Roots, barks, seeds; not for photolabile compounds
Hot air / tray drying
Usually 40–60 °C
Most drugs; the temperature must remain below the
degradation point of the marker
Vacuum drying
Reduced pressure, low temperature
Thermolabile compounds
Freeze drying
Frozen state under vacuum
Highly sensitive constituents, fresh-plant extracts
iii.
Size reduction
Grinding increases the surface area available to the solvent and ruptures cell walls, so the
diffusion path becomes shorter. The particle size should be selected with care:
•
Too coarse: incomplete extraction because diffusion from the core of the particle is
slow.
•
Too fine: the powder packs tightly, so the percolator channels and filtration is
difficult; more cell debris and colloidal matter are released, which lowers selectivity
and can foam or emulsify during partitioning.
•
Typical practice is moderately coarse powder (about 20–60 mesh, roughly 0.25–0.85
mm) for percolation and maceration, and finer powder for ultrasound, microwave and
pressurised methods where contact is enhanced by other means.
•
Heat generated during milling can degrade volatile and thermolabile compounds;
cryogenic milling is used for sensitive materials.
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iv.
Pre-treatments
•
Defatting: lipids, waxes and chlorophyll are removed by pre-extraction with a nonpolar solvent such as n-hexane or petroleum ether. This is essential before extracting
polar compounds (flavonoids, saponins, alkaloid salts) from oily seeds.
•
Enzyme inactivation: brief blanching or ethanol treatment prevents unwanted
hydrolysis of glycosides during extraction.
•
Controlled hydrolysis: acid or enzymatic hydrolysis is deliberately used when the
aglycone is the target, for example, to convert saponins to sapogenins or flavonoid
glycosides to aglycones.
•
Alkalinization: for alkaloid drugs, moistening the powder with dilute ammonia or
lime liberates free bases from their salts so they dissolve in organic solvents.
•
Swelling or moistening: allows the solvent to penetrate the cellular matrix, especially
in resinous and gummy drugs.
Principles of Extraction
i. Mechanism of solid-liquid extraction
Extraction of plant material proceeds in four overlapping steps:
1. Penetration of the solvent into the solid plant matrix.
2. Solubilization (dissolution) of the solutes into the solvent.
3. Diffusion of the dissolved solute out of the matrix (internal mass transfer), driven by the
concentration gradient.
4. Collection of the extract, that is transfer from the particle surface into the bulk solvent
(external mass transfer).
The rate of diffusion can be described qualitatively by Fick's law: the flux increases with the
concentration gradient, the surface area and the diffusion coefficient, and decreases with the length of
the diffusion path. The practical consequence is that continuously refreshing the solvent (as in
percolation and Soxhlet) keeps the gradient high and increases exhaustion of the drug.
Factors influencing extraction efficiency
Factor
Effect and practical guidance
Nature of solvent
Polarity, selectivity, viscosity, boiling point and safety determine which
compounds are dissolved (see section 5).
Particle size
Smaller particles give a larger surface area and shorter diffusion path, but
excessively fine powder causes clogging and co-extraction of impurities.
Temperature
Higher temperature increases solubility and diffusion and lowers viscosity, but
may degrade thermolabile compounds, extract more impurities and cause solvent
loss. Most flavonoid and alkaloid extractions are run in a range of roughly 40–80
°C.
Time
Yield rises rapidly at first and then plateaus once equilibrium is approached.
Prolonged extraction only adds degradation and impurities.
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Factor
Effect and practical guidance
Solvent-to-solid ratio
A larger ratio increases the gradient and yield but raises solvent cost and
concentration time. Ratios between 1:10 and 1:30 (w/v) are common in laboratory
practice.
Agitation
Stirring, shaking or circulation thins the boundary layer around particles and
speeds external mass transfer.
pH
Controls the ionization state of acids and bases. Alkaloids are soluble in organic
solvents as free bases and in water as salts; phenolic acids are extracted more
easily at acidic pH.
Pressure
Elevated pressure keeps solvents liquid above their boiling point and forces
solvent into pores (pressurized liquid extraction, supercritical extraction).
Number of extraction
cycles
Several successive extractions with smaller portions of solvent exhaust the drug
better than one extraction with the total volume.
Moisture in the drug
Some water swells the matrix and helps polar solvent penetrate; excess water
dilutes organic solvents and reduces their selectivity.
Solvent Selection
The guiding principle is 'like dissolves like'. Polar compounds dissolve in polar solvents and
non-polar compounds in non-polar solvents. An ideal extraction solvent is selective for the
target, chemically inert, of low toxicity, easy to remove, non-flammable (or manageable),
inexpensive, and acceptable to regulators for the intended product.
Properties of common solvents
Solvent
Polarity
index
Boiling
point (°C)
Typical targets and remarks
Water
10.2
100
Glycosides, tannins, mucilage, polysaccharides,
saponins, alkaloid salts; risk of microbial growth
and hydrolysis; food-safe
Methanol
5.1
65
Broad range of polar to moderately polar
compounds; highly effective but toxic, so use is
limited in food and drug products
Ethanol
5.2
78
Most widely accepted; alone or with water (30–
80%) for flavonoids, glycosides, alkaloids, tannins,
curcuminoids
Acetone
5.1
56
Flavonoids, phenolics, curcuminoids; easily
removed; flammable
Ethyl acetate
4.4
77
Flavonoid aglycones, medium polarity phenolics,
some terpenoids
Chloroform
4.1
61
Alkaloid free bases, terpenoids; toxic, restricted use
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Solvent
Polarity
index
Boiling
point (°C)
Typical targets and remarks
Dichloromethane
3.1
40
Alkaloids, terpenoids, lipophilic compounds;
volatile; restricted use
Diethyl ether
2.8
35
Volatile oils, fats, resins; extremely flammable
nHexane/petroleum
ether
0.1
60–70
Fixed oils, waxes, chlorophyll, volatile oil; used for
defatting
Primary Extraction Techniques
i. Conventional methods
a. Maceration
The coarsely powdered drug is kept in contact with the solvent in a closed vessel at room
temperature for a defined period (usually 3–7 days) with occasional or continuous agitation.
The mixture is then strained, the marc is pressed, and the combined liquids are clarified and
filtered.
•
Advantages: simple, inexpensive, suitable for thermolabile constituents.
•
Disadvantages: long duration, incomplete exhaustion, large solvent volume, marc
retains some of the extract.
•
Variants: multiple maceration (the solvent is divided into portions and applied
successively) gives better yield; circulatory maceration uses a pump to circulate the
solvent.
b. Percolation
The moistened, uniformly packed drug in a conical percolator is continuously drained of
solvent that is added at the top. The steps are: imbibition (moistening and standing for about 4
hours), packing, maceration for around 24 hours with the outlet closed, and percolation at a
controlled rate (for example 1 mL per minute per 1000 g of drug in the pharmacopoeial
method), with the first portion of the percolate being the most concentrated (the reserved
portion). The process is continued until the drug is exhausted.
•
Advantages: continuous supply of fresh solvent, more complete than maceration.
•
Disadvantages: requires skill in uniform packing, unsuitable for very fine or gummy
powders, uses more solvent.
c. Digestion, infusion and decoction
• Digestion: maceration with gentle heat (typically 40–50 °C) for improved solubility.
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•
Infusion: fresh preparation by pouring boiling or hot water over the drug and
allowing to stand for a short time (about 15 minutes); suitable for soft, easily
extracted parts such as leaves and flowers.
•
Decoction: boiling the drug in water (about 15–20 minutes or longer) for hard, woody
or root material and heat-stable, water-soluble constituents. Not suitable for
thermolabile or volatile constituents.
d. Hot continuous extraction (Soxhlet)
The powdered drug in a porous thimble is placed in the extraction chamber. Solvent vapour
rises, condenses and fills the chamber; when the level reaches the siphon top, the extract returns
to the flask, and the cycle repeats. Fresh, hot, distilled solvent contacts the drug in each cycle,
giving efficient exhaustion with relatively little solvent.
•
Advantages: high efficiency, low solvent consumption compared with maceration,
unattended operation.
•
Disadvantages: prolonged heating of the extract in the flask can degrade thermolabile
compounds; unsuitable for heat-sensitive material; not directly scalable.
e. Reflux extraction
The drug and solvent are heated together to the solvent's boiling point in a flask fitted with a
condenser, so the vapour condenses and returns. Two to three successive refluxes of one to two
hours each are common. It is faster than maceration and is widely used in industrial batch
extractors.
ii. Distillation methods for volatile constituents
• Hydrodistillation: plant material immersed in boiling water; oil and steam are
condensed and separated (Clevenger-type apparatus for laboratory work).
•
Steam distillation: live steam passes through the plant material; less risk of charring.
•
Water and steam distillation: material rests on a perforated grid above boiling
water.
•
These methods rely on the fact that the mixture of immiscible water and oil boils
below 100 °C, protecting heat-sensitive oil components.
Modern (assisted) extraction methods
Ultrasound-assisted extraction (UAE)
Ultrasonic waves (typically 20–100 kHz) produce acoustic cavitation. The collapse of
cavitation bubbles near the plant surface causes micro-jets and shock waves that disrupt cell
walls and enhance solvent penetration and mass transfer. Typical extraction times are 10–60
minutes at moderate temperature. Advantages include shorter time, lower solvent consumption
and suitability for thermolabile compounds; limitations include difficult scale-up, probe
erosion, and possible free radical formation at high power.
Microwave-assisted extraction (MAE)
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Microwave energy heats polar solvents and moisture inside plant cells by dipole rotation and
ionic conduction. The internal pressure rises until cells rupture and release the contents into the
solvent. Extraction is completed in minutes, with high yield and low solvent volume. The
solvent must absorb microwaves (ethanol, methanol, water and their mixtures do so; hexane
does not). Care is needed with heat-sensitive compounds and pressure safety.
Supercritical fluid extraction (SFE)
A fluid above its critical temperature and pressure has liquid-like solvating power and gas-like
diffusivity and viscosity. For carbon dioxide the critical point is about 31.1 °C and 73.8 bar
(7.38 MPa). The fluid dissolves compounds in the extractor; on reduction of pressure in the
separator, the CO₂ returns to gas and the extract precipitates solvent-free.
•
Pure scCO₂ is best for non-polar compounds (essential oils, carotenoids, lipids, some
terpenoids).
•
Polar compounds need a co-solvent (modifier) such as ethanol.
•
Selectivity can be adjusted by changing pressure and temperature, which allows
fractional extraction.
•
High capital cost, but it is a clean process, widely used for decaffeination, hop
extracts and spice extracts.
Pressurized liquid extraction (PLE, accelerated solvent extraction)
Extraction with liquid solvents at elevated temperature (about 50–200 °C) and pressure (about
10–15 MPa), which keeps solvents liquid above their boiling point. High temperature lowers
viscosity and surface tension and increases solubility, while pressure forces solvent into the
matrix. Extraction is complete in 5–20 minutes with automation.
Enzyme-assisted extraction (EAE)
Cellulase, pectinase, hemicellulase and other enzymes degrade the cell wall, which releases
bound compounds. It operates under mild conditions (temperature about 40–60 °C, controlled
pH) and is commonly used with water. Enzyme cost and the need for optimal conditions are
limitations.
Post-Extraction Handling
Filtration and clarification
•
Straining and pressing of the marc recovers the liquid held in the residue.
•
Filtration through cloth, filter paper, filter press or centrifugation removes fine suspended
material.
•
Settling and decantation help with viscous extracts. Filter aids (diatomaceous earth, celite)
improve flow.
•
Clarification by centrifugation, ultrafiltration or treatment with gelatin, bentonite or activated
carbon removes colloids, but activated carbon can adsorb desired compounds and must be
evaluated carefully.
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Concentration
Solvent is removed to produce a thick liquid or paste. Evaporation under reduced pressure at 40–60 °C
using a rotary evaporator (laboratory), falling-film or forced-circulation evaporators, or thin-film
evaporators (industry) protects thermolabile compounds by lowering the boiling point. Solvent recovery
by distillation reduces cost and environmental impact.
Drying
Method
Principle
Remarks
Vacuum tray drying
Evaporation under reduced pressure at
low temperature
Gives a brittle mass that is milled;
suitable for hygroscopic extracts
Spray drying
Atomizing the liquid into a hot air stream;
particles dry within seconds
Yields free-flowing powder; carriers
such as maltodextrin or silicon dioxide
often needed; inlet temperature
commonly 150–200 °C, but the product
remains at a lower temperature
Freeze drying
(lyophilization)
Sublimation of ice under vacuum
Best retention of heat-sensitive actives;
costly and slow
Fluid-bed drying /
granulation
Hot air fluidizes the particles
Used for granulating extracts with
excipients
Belt drying
Extract spread on a moving belt under
heated air or vacuum
Continuous industrial operation
Enrichment Strategies
Primary extraction gives a mixture that is only partially enriched. Enrichment exploits differences in
polarity, acid-base behaviour, molecular size, solubility, adsorption affinity and volatility between
the target compounds and the accompanying matrix. Usually, several methods are combined in
sequence.
Liquid-liquid partitioning (solvent fractionation)
The concentrated extract is suspended in water (or aqueous alcohol) and shaken successively with
immiscible organic solvents of increasing polarity, for instance n-hexane, then dichloromethane or
chloroform, then ethyl acetate, then n-butanol. Each layer collects compounds according to their
partition coefficient (K = concentration in organic phase / concentration in aqueous phase).
Fraction
Typically enriches
n-Hexane / petroleum ether
Lipids, waxes, chlorophyll, sterols, non-polar terpenoids
Dichloromethane /
chloroform
Alkaloid free bases, less polar terpenoids, some flavonoid aglycones
Ethyl acetate
Flavonoid aglycones and less polar glycosides, phenolic acids, coumarins
n-Butanol
Saponins, polar flavonoid glycosides, iridoid glycosides
Residual aqueous
Sugars, amino acids, polysaccharides, tannins, very polar glycosides, salts
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Practical points: emulsions can form, especially with saponins and proteins; they are broken by adding
salt, centrifuging or standing. Repeating the extraction three times with smaller volumes recovers more
solute than a single large-volume extraction.
Acid-base partitioning for alkaloids
This is the classic and most effective enrichment method for basic nitrogen compounds.
1. Extract the plant material with acidified water or aqueous alcohol (for example 1–5%
hydrochloric or sulfuric or tartaric acid). Alkaloids form salts and dissolve.
2. Wash the acidic solution with a non-polar solvent (hexane or ether) to remove neutral and
acidic lipophilic impurities, which are discarded.
3. Basify the aqueous phase with ammonia or sodium carbonate to about pH 9–10. Alkaloid
salts convert to free bases, which are now insoluble in water.
4. Extract the basic solution with chloroform or dichloromethane (or ethyl acetate). Alkaloid
bases move into the organic layer.
5. Wash, dry over anhydrous sodium sulfate and evaporate to give a total alkaloid fraction, often
at 5–10 times or higher enrichment.
A similar principle is used for acidic compounds (phenolic acids, some lignans): extraction at alkaline
pH into water, washing with organic solvent, acidification, and re-extraction into an organic solvent.
Adsorption and macroporous resin chromatography
Macroporous adsorption resins (non-polar polystyrene-divinylbenzene types such as the D101 and
Amberlite XAD series, or weakly polar and polar types) are the most widely used industrial method for
enriching flavonoids, saponins, phenolics, anthocyanins and glycosides from aqueous or hydroalcoholic
extracts.
1. Resin pre-treatment: wash with ethanol and then water to remove monomers and porogens.
2. Loading: pass the crude aqueous extract through the resin column at a controlled flow
(typically a few bed volumes per hour). Target compounds adsorb through hydrophobic and
hydrogen-bond interactions; sugars, salts and very polar impurities pass through.
3. Washing: water (2–5 bed volumes) removes polar impurities.
4. Elution: stepwise gradient of ethanol in water (for example 30%, 50%, 70%, 95%). The
fraction with the target compound is collected.
5. Regeneration: flush with higher ethanol concentration, and if necessary dilute acid or alkali,
then water.
Selection depends on the resin's surface area, pore diameter, polarity and the molecular size and polarity
of the target. Enrichment factors of 5- to 20-fold in total flavonoids or saponins with recoveries above
80% are commonly reported. The technique is scalable, uses relatively cheap ethanol and can be run
continuously.
Precipitation and crystallization
•
Alcohol precipitation: adding ethanol to an aqueous concentrate to 70–80% precipitates
polysaccharides, proteins and pectins, which are filtered off. This purifies the supernatant that
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contains smaller molecules; conversely, polysaccharides themselves can be enriched from the
precipitate.
•
Water precipitation: pouring an ethanol extract into water precipitates resins and lipophilic
constituents; useful for removing resin or enriching such compounds.
•
Isoelectric or pH precipitation: compounds precipitate at their isoelectric point or as
insoluble salts.
•
Salting-out: ammonium sulfate or sodium chloride reduces solubility of organic compounds.
•
Crystallization and recrystallization: achieved by cooling, solvent evaporation or antisolvent addition; this is the final step to obtain high-purity enriched products such as
curcuminoids or rutin.
•
Traditional lead acetate precipitation of tannins is now avoided because of toxicity;
gelatin, polyvinylpolypyrrolidone (PVPP) or resin adsorption are used to remove tannins.
Membrane separation
Pressure-driven membranes separate molecules by size and are attractive because they operate at
ambient temperature without phase change.
Process
Approximate cut-off
Application
Microfiltration
0.1–10 µm
Removes suspended solids and microorganisms
Ultrafiltration
Roughly 1–500 kDa
Removes polysaccharides, proteins, and colloids;
clarifies extracts
Nanofiltration
Roughly 200–1000 Da
Retains small molecules such as flavonoids and saponins;
passes salts and solvent; also concentrates without
heating
Reverse osmosis
Below about 200 Da
Concentration of aqueous extracts
Fractional extraction with supercritical CO₂
By stepping pressure or by using staged separators, non-polar compounds (volatile oil, waxes and
pigments) can be fractionated in sequence, leaving an enriched residue or fraction. In turmeric, for
example, the volatile oil is first removed with pure scCO₂ and the curcuminoids are then extracted using
ethanol-modified CO₂.
III. Latest Methods of Extraction of Essential Oils
Essential oils (EOs) are complex mixtures of volatile, lipophilic secondary metabolites
produced by aromatic plants. They give plants their characteristic odour and flavour and
function in defence against herbivores and pathogens, in attracting pollinators and in
allelopathy. They are called “essential” because they carry the essence (fragrance) of the plant,
not because they are nutritionally essential.
Essential oils are obtained from flowers, leaves, bark, wood, roots, rhizomes, seeds, fruit peels
and resins. Typical examples are lavender, peppermint, eucalyptus, rose, jasmine, sandalwood,
clove, cinnamon, lemongrass, citrus peel, tea tree and palmarosa.
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Where are essential oils stored in the plant?
Oil is stored in specialised secretory structures: glandular trichomes (Lamiaceae such as mint,
basil, lavender), oil cells (Piperaceae, Lauraceae), secretory cavities or oil glands in rind
(Rutaceae, i.e. citrus), and resin ducts (Pinaceae, Apiaceae). The location and depth of these
structures directly affect which extraction method works best. Surface trichomes rupture
readily, whereas oil embedded in seeds or woody tissue requires cell-wall disruption.
Why look for new extraction methods?
Conventional hydrodistillation and steam distillation have been used for centuries, but they
have known drawbacks: long processing times (3 to 8 hours), high energy consumption,
thermal degradation of labile compounds, hydrolysis of esters, loss of polar constituents in
wastewater, and poor recovery of high-boiling components. Solvent extraction leaves residues
and may be toxic. Growing consumer demand for natural products, tighter regulation,
sustainability goals (Green Chemistry, Green Extraction principles) and the need for higherquality oil for pharmaceuticals, food, cosmetics and aromatherapy have driven the development
of newer techniques.
Conventional Methods in Brief
Hydrodistillation (HD)
Plant material is fully immersed in boiling water. Oil vapours and steam co-distil at a
temperature below the boiling point of either component alone (Dalton’s law of partial
pressures), are condensed, and the oil separates from the hydrosol in a Florentine flask. Simple,
but slow and with a risk of hydrolysis and overheating.
Steam distillation
Live steam is passed through a bed of plant material supported above the water level. It is the
most widely used industrial method because it avoids direct contact of the plant with boiling
water, giving better quality than HD.
Cold pressing
Used for citrus oils (orange, lemon, bergamot, mandarin, lime). Rind oil glands are ruptured
mechanically and the oil is recovered as an oil–water emulsion followed by centrifugation. No
heat is applied, so fresh top notes are preserved, but co-extracted waxes and pigments need to
be removed.
Solvent extraction and enfleurage
Hexane, petroleum ether or ethanol extracts flowers to give a waxy “concrete”, which is then
washed with ethanol to yield an “absolute”. Enfleurage uses purified fat to absorb fragrance
from flower petals. These methods capture delicate aromas but leave solvent traces and are
labour intensive.
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Latest Methods of Essential Oil Extraction
Microwave-Assisted Extraction
Principle
Microwaves (usually 2.45 GHz) are non-ionising electromagnetic waves that heat materials by
two mechanisms: dipole rotation of polar molecules (mainly water in the plant tissue) and
ionic conduction of dissolved ions. Because heating is volumetric and internal, the water inside
the glandular structures is heated rapidly, builds up internal pressure and ruptures the cells,
releasing oil to the surface where it is carried away by steam or solvent. Heating is therefore
from inside to outside, in contrast to conduction heating in conventional distillation (outside to
inside).
Process parameters
• Microwave power (typically 300–1000 W for lab systems): higher power speeds
extraction but may degrade oil.
•
Irradiation time: yield rises then plateaus; over-irradiation causes charring and losses.
•
Moisture content of material: water content is essential for SFME because water
absorbs microwaves; very dry material must be rehydrated.
•
Particle size and loading: influences penetration depth of microwaves and heat
uniformity.
•
Solvent dielectric properties (in solvent-based MAE): polar solvents absorb strongly,
non-polar (hexane) are microwave-transparent.
Advantages and limitations
Advantages: very short extraction time, reduced energy use (up to 70–90% less than HD in
many reports), reduced or zero solvent, higher content of oxygenated compounds, less
wastewater, and good reproducibility.
Limitations: capital cost, penetration depth limits scale-up, risk of hot spots, unsuitable for
materials with very low moisture unless rewetted, and need for safety controls (leakage,
pressure). Industrial-scale continuous microwave units are now available and remain an active
area of development.
Examples: SFME has been successfully applied to lavender, rosemary, thyme, peppermint,
orange peel, basil and cardamom, often giving oil of comparable or better quality than HD in
a fraction of the time.
Ultrasound-Assisted Extraction (UAE)
Principle
Ultrasound consists of sound waves above 20 kHz. When passed through a liquid, it produces
alternating compression and rarefaction cycles. During rarefaction, microbubbles form and
grow; during compression they collapse violently, a phenomenon called acoustic cavitation.
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Collapse near the plant surface generates micro-jets, shock waves, localised high temperatures
and pressures, causing cell wall disruption, pore formation and enhanced mass transfer of oil
into the medium (solvent, water or a hydrotrope). Additional mechanisms include
fragmentation, erosion, sonocapillary effect and sonoporation.
Equipment
Ultrasonic baths (indirect sonication, 20–40 kHz, low intensity) and probe (horn) systems
(direct sonication, higher intensity, better reproducibility). Industrial flow-through
sonoreactors are available. UAE is used either as a stand-alone extraction or as a pre-treatment
before hydrodistillation (ultrasound-assisted hydrodistillation, UAHD) or in combination with
microwaves (ultrasonic–microwave-assisted extraction, UMAE).
Parameters
• Frequency and intensity (W/cm²): lower frequencies (20–40 kHz) produce stronger
cavitation.
•
Temperature: raises vapour pressure and lowers cavitation intensity; an optimum is
required.
•
Solvent: viscosity, surface tension and vapour pressure influence bubble behaviour.
•
Time, liquid-to-solid ratio and particle size.
Advantages: simple, cheap, low temperature, short time, applicable to a wide variety of
materials, low solvent consumption. Limitations: free radical generation at high intensity may
degrade some compounds; probe erosion; scale-up requires careful reactor design; non-uniform
energy distribution in baths.
Supercritical Fluid Extraction (SFE), Mainly Supercritical CO2
Principle
A substance above its critical temperature and critical pressure is a supercritical fluid with
gas-like diffusivity and viscosity and liquid-like density and solvating power. For carbon
dioxide, the critical point is 31.1 °C and 73.8 bar (7.38 MPa). Small changes in pressure and
temperature markedly change the density and hence the solvent power of the fluid, so extraction
can be tuned to target specific compound classes. When pressure is released after extraction,
CO2 returns to gas and leaves the extract solvent-free.
Process description
1. Liquid CO2 from a cylinder is cooled, pressurised by a high-pressure pump and heated
to supercritical conditions.
2. Supercritical CO2 flows through the extraction vessel packed with ground plant
material and dissolves the volatile oil.
3. The CO2–oil mixture is depressurised through a back-pressure valve in one or more
separators, where the oil precipitates and CO2 is gasified.
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4. CO2 is recompressed and recycled. A co-solvent (entrainer) such as ethanol (1–10%)
may be added to raise the polarity.
Fractionation
Multi-stage separators operated at different pressures and temperatures allow fractionation of
the extract, for example separating waxes and cuticular lipids (precipitated first) from the true
volatile fraction, giving a cleaner oil than would be obtained in a single-stage separation.
Advantages: no toxic solvent residues, moderate temperature, no oxygen exposure, tunable
selectivity, oil profile closer to the natural aroma of the plant, CO2 is non-toxic, nonflammable, cheap and recyclable. Limitations: high capital and operating cost, need for highpressure equipment and trained operators, batch operation (semi-continuous), co-extraction of
waxes in some cases. It is widely used commercially for spices, hops, herbs and high-value oils
(e.g., ginger, black pepper, chamomile, rose, vanilla).
Subcritical Water Extraction (SWE) / Pressurised Hot Water Extraction
Water heated between 100 °C and 374 °C (its critical temperature) under enough pressure
(typically 10–60 bar) to remain liquid is called subcritical water. As temperature rises, the
dielectric constant of water falls sharply from about 80 at 25 °C to about 27 at 250 °C, similar
to ethanol or methanol. Hence, hot pressurised water can dissolve moderately polar and even
non-polar organic compounds without organic solvents. Viscosity and surface tension also fall,
favouring penetration into the matrix.
For essential oils, SWE is generally run at 100–200 °C, in static or dynamic mode, with extract
collected after cooling and separated from water (liquid–liquid partition or adsorption). It is
particularly effective for recovering oxygenated compounds, which are more valuable than
hydrocarbon terpenes, and gives cleaner oils with fewer terpene hydrocarbons than
conventional HD. It also suits extraction of polar aroma compounds from spices such as cumin,
fennel, coriander, caraway and clove.
Advantages: water is the only solvent (cheap, safe, green), short time, selectivity by
temperature. Limitations: thermal degradation above ~200 °C, need for pressure vessels and
corrosion-resistant materials, and the need to separate dilute oil from large volumes of water.
Pressurised Liquid Extraction (PLE) / Accelerated Solvent Extraction (ASE)
A solvent (ethanol, ethyl acetate, water or mixtures) is kept liquid above its normal boiling
point by applying pressure (typically 50–200 bar) at 40–200 °C. Elevated temperature increases
solubility and diffusion, while pressure forces solvent into pores. Extraction takes 5–20 minutes
in automated cells with much lower solvent use than Soxhlet. PLE is more common for nonvolatile extracts but is applied to aromatic materials to obtain aroma-rich extracts, particularly
where a wider range of constituents than steam-volatiles is required.
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Pulsed Electric Field (PEF) Assisted Extraction
PEF applies very short (microsecond to millisecond) high-voltage pulses (typically 0.5–10
kV/cm for plant tissue, up to 20+ kV/cm for tough material) to plant material placed between
two electrodes in a treatment chamber. The electric field induces electroporation: the
formation of pores in cell membranes when the transmembrane potential exceeds a critical
value (about 0.5–1 V). Pores increase permeability and mass transfer, allowing oil and other
metabolites to escape more easily.
PEF is a non-thermal pre-treatment, applied before hydrodistillation, pressing or solvent
extraction. It shortens the subsequent extraction time, reduces energy, and raises yield and
quality. It has been reported to enhance recovery in orange peel, lavender, rosemary, thyme,
and other materials. Advantages: minimal heating (temperature rise below 10 °C), low energy
per kg, continuous operation possible, easy scale-up in liquid streams. Limitations: equipment
cost, need for conductive medium (water), less effective for dry, non-conductive material, and
treatment parameters (field strength, pulse number, specific energy) must be optimised for each
plant.
Enzyme-Assisted Extraction (EAE)
Plant cell walls are composed of cellulose, hemicellulose, pectin and lignin, which act as a
barrier to oil release, especially in seeds, roots and woody tissues. Enzymes such as cellulase,
pectinase, hemicellulase, xylanase, β-glucosidase and protease selectively hydrolyse these
polymers, weakening the wall and freeing oil and bound aroma precursors. Enzymes can also
release glycosidically bound volatiles, increasing aroma yield.
Procedure: powdered or macerated material is incubated in buffer at optimum pH (usually 4–
6) and temperature (40–55 °C) with enzyme (0.5–5% w/w) for 1–24 h, followed by
conventional distillation or extraction. EAE has been applied to cardamom, ginger, turmeric,
fennel, caraway, cumin, rose petals, and citrus peel.
Advantages: mild conditions, specific action, environmentally friendly, higher yield and
improved quality. Limitations: enzyme cost, sensitivity to pH, temperature and inhibitors,
longer incubation time compared with physical methods, and difficult scale-up. Combination
approaches (enzyme + ultrasound or enzyme + microwave) are common.
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