Transcript 434- Part II [Lec-2

434 PHG
Recent Approaches in
Medicinal Plants Analyses
Prof. Mohammed Abdulaziz Al-Yahya
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Phytochemical Study
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To carry phytochemical study the following points must
be fulfilled:
1- Selection of promising plant materials.
2- Proper collection of selected plants.
3- Authentication of plant material.
4- Drying of plant materials.
5- Garbling of the dried plants
6- Packing, storage and preservation
7- Grinding of the dried plants.
8- Extraction and fractionation of constituents.
9- Methods of separation and purification.
10- Methods of identification of isolated compounds
(structure elucidation e.g.: UV, IR, MS, 1H-NMR and 13C-NMR).
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1. SELECTION OF PROMISING PLANT MATERIAL
The choice of promising plant depends upon the following:
1- A plant which has a biological activity.
2- A plant used in folk medicine.
3- A plant which shows a particular toxicity.
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2. Proper collection of selected plants
Drug may be collected from:
1- Wild plants.
Wild plant
2- Cultivated plants.
Cultivated plant
Disadvantage
Advantage
1- Scattered in large or
unlimited area
Present in limited area.
2- Difficult to reach
Easy to reach
3- The collector must be
highly skilled botanists
The collector must not be
skillful person
Continuous supply
4- Deficiency may occur due
to continuous collection
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Rules for collection

The material is best collected when the organ in question
has reached its optimal state of development:
1- Roots and rhizomes are collected at the end of the
vegetation period, i.e. usually in the autumn.
2- Bark is collected in the spring.
3- Leaves and herbs are collected at the flowering stage.
4- Flowers are usually gathered when fully developed.
5- Fruits and seeds are collected when fully ripe.
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The following precautions should be considered during
stage of collection:
a- The proper time of the day, time of the year and
maturity stage of collection is particularly important
because the nature and quantity of constituents may
vary greatly in some species according to the season
and time of collection.
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●The most advantages time of collection is when the plant
containing the active principals is highest in its content,
example:
1- Time of year
e.g. Hyoscyamous contain less amount of alkaloids in
winter than in summer.
2- Time of the day
e.g.1 Cardiac glycoside in Digitalis leaves are in higher
amount at afternoon than in the morning.
e.g.2 Solanaceous plants have higher quantities of alkaloids
in the morning than in the afternoon.
3- Stage of maturity
e.g. Solanaceous plants have higher quantities of alkaloids
when collected in the flowering stage.
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b- The collected plant should be free from any contamination.
The main causes of contamination are:
i- Collection of mixtures of plants by error.
ii- Collection of closely similar species growing side by
side and incorrectly assumed to be the same.
iii- Collection of plants which have a parasite within it.
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c- Collecting plants which are free from diseases (i.e. which
are not affected by viral, bacterial, fungal infection).
This may cause:
i- Infection may seriously alter plant metabolism and
unexpected products could be formed possibly in large
amounts (causing confusion).
ii- Infection may cause the presence of products of
microbial synthesis (causing confusion).
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3. Authentication of plant material
This may be confirmed by:
1- Establishing the identity by a taxonomy experts.
2- Collection of a common species in their expected habitat by
a field botanist.
3- By comparing the collecting plant with a voucher specimen
(herbarium sheet)
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4. Drying of plant materials
Drying
The most common method for preserving plant
material is drying.
- Enzymatic processes take place in aqueous solution.
Rapid removal of the water from the cell will,
therefore, largely prevent degradation of the cell
constituents.
- Drying also decreases the risk of external attack, e.g.
by moulds.
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Note: Drying should be carried out as quickly as possible
without using high temperatures to prevent chemical
changes of thermo-labile constituents e.g. volatile oils.
Living plant material has a high water content:
* leaves may contain 60-90% water.
* Roots and rhizomes 70-85%.
* Wood 40-50%.
* The lowest percentage is found in seeds, often no
more than 5-10%.
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Aim of drying:
1- Ease of transport.
2- Ease of grinding
3- Inhibit the growth of microorganisms.
4- Preservative of active constituents.
Drying is done in:
-Shade and in sunlight (Natural drying).
- Hot air drying or by freeze-drying (Artificial drying).
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Changes may occur during the drying:
1- Size and weight:
Drug when drying will be smaller in size and lose 80-90 %
of their original weight.
2- Shape and appearance:
Black pepper on drying show polygonal reticulation (due
to presence of stone cell in the hypodermis)
3- Color:
Tea leaves change from green to dark brown, almost black.
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Freeze-drying
• Freeze-drying (Lyophilization) is a very mild method.
• Frozen material is placed in an evacuated apparatus
which has a cold surface maintained at -60 to -80
°C. Water vapor from the frozen material then
passes rapidly to the cold surface.
• The method requires a relatively complicated
apparatus and is much more expensive than hot-air
drying. For this reason, it is not used as a routine
method, but it is very important for drying heatsensitive substances, e.g. antibiotics and proteins.
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4- Odor:
Vanilla pods odorless when fresh and on drying acquire
a fragrant, pleasant aromatic odor due to liberation of
vanillin which has a charr. or nice odor.
5- Active constituent:
Slow drying of vanilla pods lead to obtain vanillin from
glucovanillic alcohol.
vanillin
Glucovanillin
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How drying can change the yield
Fresh Willow (Salix) Leaves
Vaccuum Dried
Bad yield of Simple
Phenolic Glycosides
Good yield of
Condensed Tannins
(Polyphenols)
Air Dried
Good yield of Simple
Phenolic Glycosides
Bad yield of
Condensed Tanins
(Polyphenols)
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5. Garbling of the dried plants
 Garbling is the final step in the preparation of a crude
drug.
 Garbling consists of the removal of extraneous matter,
such as other parts of the plant, dirt and added adulterants.
Excessive dust can clog percolators and result in a turbid
extract which is hard to clarify.
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6. Packing, storage and preservation

Drugs with essential oils deteriorate quickly through
evaporation, oxidation and polymerization of the
substances constituting the essential oil.

Tannins on the other hand, have an almost unlimited
durability.
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7. Grinding of the dried plants

The plant is ground into small particle size to
facilitate extraction.

Large particles take a longer time for complete
extraction than small ones.
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In order to keep crude drugs as long as possible:
1. It is essential to store them in a dry condition in carefully
closed containers.
2. It is also advisable to store them in light resistant
containers such as, tin cans, amber glass container.
because - even if light does not affect the active constituents
it almost causes changes in the appearance of the drug,
especially loss of color.
3. It is also necessary to protect the drug against insect attack.
4. Drugs must always be stored at as low temperature as
possible because high temperature accelerate all chemical
reactions.
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8- EFFECTIVE EXTRACTION
There is no general (universal) method for the extraction
of plant materials.
The precise mode of extraction depends on:
1- The texture of the plant material.
2- The water content of the plant material.
3- The type of substances to be extracted or nature of
active constituents.
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Extraction: is the separation of medicinally active portion of
plants or animal tissues through the use of selective
solvent and suitable methods of extraction.
The principal methods of extraction are:
A- Maceration
B- Infusion
C- Percolation
D- Decoction
E- Digestion
F- Continuous hot extraction technique (Soxhlet extraction
process).
G- Liquid-liquid extraction
H- Solvent-solvent ppt.
I- Distillation
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A-Maceration:
- This method is used for cold water soluble active constituents.
- It consists of macerating the plant material in cold water (15-20
°C) for several hours. e.g. liquorice.
B- Infusion
- Infusion is used for water-soluble and easily extracts principles.
- The plant material is placed in a pot and wetted with cold water.
Immediately afterwards, boiling water is poured over it, then left
to stand, covered with a lid for about 15 min after which the tea is
poured off. e.g. herbal tea.
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C- Percolation:
The plant material is placed in percolator and macerated with
the solvent for several hours, continuous feeding of solvent until
complete extraction is occur.
Principle of action:
- The instrument used to hold the powder is called a percolator.
- The liquid coming from the percolator impregnated with the
soluble constituents is called the percolate.
- The residual drug remaining in the percolator after the
extraction of the soluble constituents is called the marc.
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D- Decoction
- It was used for water soluble and heat stable constituents.
- The method involves boiling the drug with water for 10 min,
then allowing it to cool to about 40°C.
E- Digestion
- This method is suitable for hard barks or woods which are
difficult for water to penetrates.
- Digestion is also considered as macerated but at relatively
elevated temperature 35-40C but not exceed 50C.
e.g. cinnamon.
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F-Continuous hot extraction technique (Soxhlet extraction
process)
-This procedure is the classical chemical and commonest
method of extraction of organic constituents.
-The powdered material is continuous extracted
successively in a Soxhlet apparatus with a range of
solvents of increasing polarity.
(Starting with least polar solvent and ending with the most
polar one:
Petroleum ether then chloroform then ethyl acetate
then, methanol and finally water).
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Extraction with each solvent is continued until side tube of Soxhlet is colorless.
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G- Liquid-liquid extraction:
• In this technique, the solute molecules are partitioned between
two immiscible solvents.
• The amount of solute in each phase will depend upon the relative
solubility in each solvent which in turn is related to their polarity.
• It is measured by the partition coefficient (K) which is constant.
Partition coefficient (K) = mole fraction of solute in phase 1 (upper phase)
mole fraction of solute in phase 2 (lower phase)
• The success of this method depends upon the selectivity of the
solvents for the required compound.
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H-Solvent-solvent precipitation:
 The extract dissolved in a suitable solvent, is mixed
with a less polar miscible solvent causing the selective
precipitation of the less soluble plant constituents.
e.g. Precipitation of triterpenoid saponins from
methanol extract of Phytolacea dodecandra by
the addition of acetone.
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I- Distillation Methods:
• There are two types of traps:
One for oils lighter than water and the other for oils heavier
than water.
• These two types differ only in the mechanism of the return
of the aqueous layer to the distillation flask, keeping the
volatile oil layer in its position.
Types of distillation are used:
1-Water and steam distillation
2-Direct steam distillation
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Points for consideration in the distillation method:
1- It is often necessary to subject the plant material to special
treatment prior to steam distillation e.g. cut or crushed.
Crushing or cutting facilitates penetration of water into
oil- containing structures in the plant.
e.g. Oil cells, glandular hairs.
2- For removal of water or moisture which might be present in
the prepared volatile oil, anhydrous sodium sulfate is usually
used.
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Choice of solvents
1.
2.
3.
4.
5.
6.
Be highly selective for the compound to be extracted.
Have a high capacity for extraction.
Not react with the extracted compound.
Have a low price.
Be harmless to man and to the environment.
Be completely volatile.
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
According to the pharmacopoeias, ethanol is the solvent
of choice for obtaining classic extracts.

The ethanol is usually mixed with water to induce
swelling of the plant particles and to increase the porosity
of the cell walls which facilitates the diffusion of
extracted substances from inside the cells to the
surrounding solvent.
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As a general empirical rule:
 Non polar solvents (petroleum ether and hexane)
will dissolve non-polar compounds (fats and waxes).
While polar solvents (methanol, ethanol and water)
dissolve polar compound (alkaloid salts and sugars).
(Like dissolve like)
 The affinity of the solute for the organic phase may
be greatly increased by using mixture of solvents
instead of single ones (used mixtures of solvent to
increase the solubility).
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Example: solublization of an aliphatic carboxylic acid in
ethanol, acetone and a mixture of both.
O------------H-O-CH
2-CH3
In ethanol -R-C
Hydrogen bond
O-H
O
In acetone R-C
Hydrogen bond
CH3
O-H---------O=C
CH3
In a mixture of acetone and ethanol
Increase solubility of carboxylic acid by
addition of ethanol and acetone.
And the solubility increased due to the
formation of hydrogen bonds.
O
HO-C 2H5 (ethanol)
CH3
R-C
OH
O=C
(acetone)
CH3
Fresh Leaves or Flowers
Homogenize for 5 min in MeOH-H2O (4:1) (10 x Vol or Wt.), Filter
Filterate
Residue
Extract with EtOAc
(x5), Filter
Residue
Fibre (mainly
Polysaccharides)
Filterate
Evaporate to 1/10 vol (<40 ºC)
Acidify to 2M H2SO4
Extract with CHCl3 (x3)
Aqueous acid layer
CHCl3 Extract
Dry, Evaporate
Evaporate
Neutral Extract
Moderately Polar Extracts
(Terpenoids & Phenolics)
Basify to pH
10 with NH4OH,
Extract with
CHCl3-MeOH
(3:1, twice)
(Fats & Waxes)
CHCl3-MeOH Extract
Dry, evaporate
Basic Extract
(Most Alkaloids)
Aqueous basic layer
Evaporate ,
extract
with MeOH
Methanol Extract is Polar
(Quaternary alkaloids & N-oxides)
General Procedure for extracting fresh plant tissues and fractionating
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into different classes according to Polarity