Unit- V
Basics of Phytochemistry
Modern methods of extraction
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Natural Product Research
Scientists are engaged in investigating
plants, microorganisms, marine
creatures and many other forms of life
for biological activity.
2
Plant based products
Since ancient time plant based products have been
investigated extensively and biologically useful chemicals
were isolated from them, some of which are being used in
modern medicine.
Search is continuing for newer and newer plant materials
and their interaction with biological system. Whenever
such plant material is found to be useful, it is taken up for
further investigation, as regard to constituents present for
its biological activity. On confirmation of its biological
activity, the suitable extracts or isolated Phytoconstituents
are prepared from the plant material and put into usage.
3
Natural Product vs. synthetic chemicals
The general public has
developed somewhat
irrational suspicion of the
safety and desirability of
synthetic chemicals and
has viewed naturally-
derived substances and
extracts as being
inherently safer and more
desirable.
4
Management of health
Today many consumers prefer to treat
themselves with phyto-pharmaceuticals
or herbal preparations and the sale of
these are increasing steadily each year
in most first world countries.
5
Development in sector
This has led to the
development of new field called
Herbal Drug Extraction. Efforts
are being made by individual
manufacturers to improve yield,
as well as composition of total
extracts and also of phyto-
constituents of interest,
wherever desired.
6
Active constituents
It was found in many cases the activity of the crude plant extract could
be reproduced by an equivalent dose of the isolated ‘active constituent’
and this has laid the foundation for modern scientific presentation of
orthodox pharmaceuticals. These active constituents of one molecule
type, ‘the single chemical entity’, could either be isolated from natural
sources, or synthesized and incorporated into dosage form.
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Need for extraction and isolation
To formulate into suitable dosage form
To remove unwanted material/constituents
To reduce the dose of drug /make it more potent
For evaluation and exact dose determination
For development of suitable derivatives which
are more potent
Extractions are carried out for variety of reasons
: to get concentrate of actives, to purify the
compounds for further processing, to isolate the
compound for characterization, etc.
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Reasons for undertaking
extraction and isolation of actives
For evaluation and
exact dose
determination
Vasicine
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Artemisinin and its Analogues
Artemisinin is not very soluble either in
water or oil. This and its short elimination
half life led to the search for the
derivatives that had improved
pharmacological properties as well as
better antimalarial activity
Artemisinin may be reduced to the lactol
(hemiacetal) dihydroartemisinin and this
has been used for the semisynthesis of
range of analogues :
Artesunate (water-soluble: for oral, rectal,
intramuscular, or intravenous use)
Artemether (lipid-soluble: for oral, rectal
or intramuscular use)
Arteether, Artelinic acid Artelinic acid
Arteannuic acid
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Quinine and synthetic analogues
11
Pyrethroids and synthetic
analogues
Transfluthrin
Allethrin I (R = −CH3)
Allethrin II (R = −COOCH3)
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Barbaloin to Diacerein
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Extraction
Extractions are very common and familiar to
us.
Living organism consists of complex
mixtures of chemicals, usually held within
cellular structural materials (mostly protein,
lipid and polysaccharides) Some are desired
while others are not. This initial step in
separating part from whole can be referred
to, as ‘extraction’ and number of methods
exist for this purpose.
Thus extraction is the process of moving
one or more compounds from one phase to
another.
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Properties of compounds to be extracted
Polarity
Effect of varying pH
Thermo stability
Properties of solvent/s to be used
Like dissolve like, boiling point, flammability, toxicity,
reactivity, presence of additives, cost, etc.
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Major constituents of medicinal plants
Majority of the constituents of medicinal plants, which are
therapeutically active, are secondary metabolites and they can be
conveniently grouped in the following classes as per their
chemical nature. It is important to understand the properties of
these commonly encountered classes of compounds, which will
be useful in selecting right combination of parameters for their
effective extraction.
Carotenoids Volatile oils/Essential oils
Alkaloids Glycosides
Saponins Phenolic Compounds
Tannins Flavonoids
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Types of herbal extracts
Herbal extracts are preparations obtained by extracting herbal drugs
with suitable extraction medium (menstrum).
1. Aqueous drug extracts (mainly obtained by (decoction, infusion and
maceration methods)
2. Tinctures : are prepared by using varying concentration of ethanol
3. Fluid extracts
4. Thin extracts
5. Thick extracts or viscous extracts
6. Dry extracts
7. Oily drug extracts
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Steps involved in extraction process
1. Comminution / Pulverisation
2. Extraction
3. Filtration of extract
4. Concentration of extract
5. Drying of extracts
6. Pulverisation of dry extracts
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Equipments for size reduction
Cutting mill
Hammer mill
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Filtration
Centrifuge
Nutch filtration
Filter press
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Evaporation/concentration
Wiped film evaporator
Agitated thin film evaporator
Falling film evaporator
Rising film evaporator
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Solvent recovery
22
Drying of extracts
Rotary vacuum dryer
Spray dryer
Tray dryer
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Methods of extractions
Maceration
Digestion
Percolation
Decoction
Infusion
Refluxation
Soxhlation
Sonication
Enzymatic pretreatment
Accelerated solvent extraction (pressurized
fluid extraction) ASE
Microwave assisted extraction (MAE)
Supercritical fluid extraction
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Maceration : Maceration consists of extraction by
soaking the whole or coarsely powdered crude
drug in the menstrum until the cellular structure
is thoroughly penetrated and the soluble portions
are softened and dissolved. Diffusion may be
assisted by shaking or by stirring. The mixture
then is strained, the marc (the damp solid
material) is pressed, and the combined liquids are
clarified by filtration or decantation after
standing. Extraction should be carried out in a
closed vessel to prevent evaporation of solvent.
The process of maceration is well suited for
extraction of crude drugs containing little or no
soft tissue. Maceration is usually conducted at a
temperature of 15-200 and for 2-7 days. Due to the
difficulty of extracting completely in some cases,
repeated maceration is performed.
Digestion : This is a form of maceration in which gentle
heat is used during the process of extraction. It is used
when moderately elevated temperature is not
objectionable. The solvent efficiency of the menstruum
is thereby increased. 25
Decoction : In this process, the crude drug is boiled in a specified volume of
water for a defined time; it is then cooled and strained or filtered. This
procedure is suitable for extracting water-soluble, heat stable constituents.
This process is typically used in preparation of Ayurvedic extracts called
“quath” or “kawath”. The starting ratio of crude drug to water is fixed, e.g. 1:4
or 1:16; the volume is then brought down to one-fourth its original volume by
boiling during the extraction procedure. Then, the concentrated extract is
filtered and used as such or processed further.
26
Refluxation : The material to be extracted is refluxed with solvent. This
is done to happen the chemical reaction, e.g. hydrolysis of glycosides.
when continuous heat may be desirable in order to increase extraction.
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Enzymatic pretreatment : In plants,
the phytoconstituents of interest are
present in a specially organized
matrix or tissue, which is made up of
complex polymeric network (
proteinaceous and polasaccharide).
Thus partial or complete destruction
of this polymeric network , which can
be carried out by the use of specific
enzymes (like cellulase, amylase,
pectinase, lipase, tannase, etc.)will
facilitate the release of
phytoconstituents in the surrounding
solvent.
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Separation of volatile oil
Enfleurage method
Ecuelle method
Steam Distillation Clavenger Apparatus
Soxhlet extraction process
Introduction
• A Soxhlet extractor is a piece of laboratory apparatus invented
in 1879 by Franz von Soxhlet. It was originally designed for
the extraction of a lipid from a solid material.
Advantages
• it has the advantages of small solvent
dosage,
• high efficiency and
• complete extraction.
Infusion : is the process of extracting chemical compounds or
flavors from plant material in a solvent such as
water, oil or alcohol, by allowing the material to remain
suspended in the solvent over time (a process often
called steeping). An infusion is also the name for the
resultant liquid. The process of infusion is distinct
from decoction, which involves boiling the plant material.
Infusion is a very simple chemical process used
with botanicals that are volatile and dissolve readily, or release
their active ingredients easily, in water, oil or alcohol. The
botanicals are typically dried herbs, flowers or berries. The
liquid is typically boiled (or brought to another appropriate
temperature) and then poured over the herb, which is then
allowed to steep in the liquid for a period of time (for not more
than 15 to 30 minutes). The liquid may then be strained or the
herbs otherwise removed from the liquid. Unless the infusion
is to be consumed immediately, it may then be bottled and
refrigerated for future use. Quantities of the herb and liquid
used will vary according to the herb or how strong the infusion
is required to be. A common proportion used is 28 g (one
ounce) of herb to 0.5 L (one pint) of liquid.
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Accelerated solvent extraction (Pressurized
fluid extraction) : In this process , sample is
placed into a sealed container and solvent is
pumped through this extraction vessel.
Because a modest pressure is applied,
temperature much greater than atmospheric
boiling point can be used with liquid
extraction solvents. The technique is
automated. The application of temperature
greatly enhances solute solubility, diffusion
resulting in extractions that are qualitatively
and quantitatively equivalent to Soxhlet in
minutes, instead of hours and with
significant less solvent. Standard ASE
conaditions 100 0C, 1500 psi (103 bar)
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Supercritical fluid extraction
The traditional procedures used to isolate the components of
interest from biological tissue : include solvent maceration and
percolation, steam-distillation and hot continuous solvent extraction
by Soxhlet apparatus.
Drawbacks :
Time consuming (several hours to days)
Loss or degradation of the components specially thermolabile
constituents.
Oxidation and hydrolysis can also occur
Dilute extracts so obtained need further concentration and
purification
37
Supercritical Fluid Extraction (SFE)
separating technique is based upon the
enhanced solvating power of certain gases
above their critical point. Above the critical
temperature and pressure, the substance
becomes a supercritical fluid and
possesses the properties of gas as well as
liquid. Thus the confined gas-like mass
transfer and liquid like solvating power of
supercritical fluids is the main criteria of
their use for extraction.
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States of matter and Supercritical fluid
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Supercritical fluid extraction - Advantages
Fast extraction (20 - 60 min.)
The solvent strength of the supercritical fluid depends upon pressure
and temperature used for extraction. At a constant temperature at
lower pressure will favour non-polar analytes and extraction at higher
pressure will favour more polar and high moleculer wt. analytes.
Most of the supercritical fluids are gases at ambient and hence their
removal is greatly simplified.
SF are relatively inert, pure, non-toxic and inexpensive. Also suitable
for thermolabile compounds. Does not favour oxidation, hydrolysis or
other chemcial reactions.
Being closed system waste generation is very minimum.
Selectivity of extraction.
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Fluid Critical Temperature Critical Pressure Pc (atm)
0C(T )
c
Ethylene 9.4 50.4
Trimethoflurane 26.3 48.6
Chlorotrifluoromethane 29 38.7
Carbon dioxide 31.1 73.8
Ethane 32.4 48.8
Propylene 91.9 46
Propane 96.8 42.5
Ammonia 132.5 113.5
Water 374.1 221.2
N-Pentane 196.7 33.7
Trichloromethane 198.2 44.1
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Percolation (Continuous hot
percolation OR Soxhlation) :
In percolation raw material is
packed into a column and the
solvent is allowed to trickle
through it. Different physical
forces such as gravitation,
viscosity, adhesion, friction,
osmosis, capillarity, surface
tension and solution play an
important part in percolation.
Continuous hot percolation is
effected by the use of Soxhlet
extractor, whereby hot liquid is
made to percolate again and
again through the drug until it is
completely exhausted.
42
Microwave assisted extraction
43
Microwaves cause ions dissolved in the
microwaved sample to oscillate, colliding
with neighboring molecules or atoms.
These collisions cause agitation, motion or
heat.
44
Sonication : When sonicating liquids at high
intensities, the sound waves that propagate into the
liquid media result in alternating high-pressure
(compression) and low-pressure (rarefaction)
cycles, with rates depending on the frequency.
During the low-pressure cycle, high-intensity
ultrasonic waves create small vacuum bubbles or
voids in the liquid. When the bubbles attain a
volume at which they can no longer absorb energy,
they collapse violently during a high-pressure cycle.
This phenomenon is termed cavitation. During the
implosion very high temperatures (approx. 5,000K)
and pressures (approx. 2,000atm) are reached
locally. The implosion of the cavitation bubble also
results in liquid jets of up to 150m/s velocity. The
resulting shear forces break the cell envelope
mechanically and improve material transfer.
Instruments are available with 20 Khz and with
power of 500 w to 1 kw.
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