Extraction & isolation method of phyto constituents (individual)

(I) ALKALOIDS
Means 'alkali like' hence resembles some characters like occurring complex
amines i.e. containing one or more nitrogen atom.

Method -1:
Powdered drug
Defatted, moistened & render alkaline with Na2Co3, NH3

(Alkaloids are freed as bases)

Extract with org solvent eg. CHCl3, ether or methylene dichloride

Total Extract
Concentrate & shake well with succesive quantities of inorganic
acid.

Residual organic fraction aqueous acid solution

(Like pigment, fats, very weak
bases or CHCl3 soluble alkaloid Sulphates)
(Alkaloidal salts)
make alkaline & extract
Org solution of alkaloidal bases alkaloids with immiscible
remove solvent solvents

(Crude alkaloid mixture)

Purification by fractional Residual aquendes

crystallization Chromatographic fraction
seperation etc.

Structure identification by modern

analytical techniques like U.V.,
I.R, NMR, mass spectroscopy etc.
Method :-2

Plant material
Ethanol

Ethanol extract
Treated with 2% HCL at room temp

Aqueous fraction Residue

Diethylether

Diethylether extract Aqueous extract

Flavonoids Basify with NH3
Extract with CHCl3

CHCl3 extract Alkaline soln

Alkaloid

treated with Mayer's

Ba [OH]2 reagent
filter (Mercuric chloride)
+
KI + H20)
Sapoins & glycosides
Alkaloid Treated with
HCl

Aqueous acidic soln

PPT
Alkaloid

Method :-3
 Stass otto process
(Mostly use & easier)

Powdered crude drug

moistened with H2O
+ treat with lime
Alkaloidal freebases
(From their acid salts)
CH Cl3/ether

Organic solvent layer aqueous layer

(Concentrated) (Alkaloidal salt)
Aqueous acid NH3 or
aq KOH
Alkaloid Liberate freebase
Org. solvent

Organic extract
(Concentrated)
Drying

Total alkaloid

Method - 4
Manske's Process

Powdered Crude drug

Defatted with pet ether

Residue
CH3O H
 Alcoholic extract
Dissolve in Concentrated
water
& adjust PH2
Extract
Steam distillation

Dark sol
or

In Refrigerator heated with paraffin

Several days (to remove impurities)
(to remove impurities)

Filtrate Filtrate
ether/CHCl3 ether/CHCl3

Remove non-alkaloidal To remove non alkaloidal impurities

Impurities
Remove non alkaloidal impurities

Basified Treat with Baxified Treat with

CHCl3 or ether CHCl3 or ether
organic layer organic layer
Evaporated evaporated

Crude alkaloid Crude alkaloid

(II) GLYCOSIDES
"Organic compound from plant or animal sources which on enzymatic or acid
hydrolysis give one or more sugar moieties along with non-sugar moiety. The former
called glycone & the later as aglycone or genin".

Method - 1:

Air dried plant material

 Methanol (MeOH)

Methanol extract into water

Insoluble Soluble
Extracted
n- butanol
n -butanol extract
concetrated & ppt
with acetone
ppt contains mixture of Glycoside

Method - 2:

Fresh plant material

shake with n-butanol Shake with water
and ethylacetate (1:1)

Org-phase Aq. Phase

extract with evaporated extract with
ethylacetate invaccum n- butanol

Ethyl acetate Ethylacetate n-butanol

(insoluble) extract
SiO2 column SiO2 column

Glycoside Glycoside

Method - 3 : Stass Otto method

Powdered drug
For thermolabile Soxhlation with alcohol
drugs below 450 C
Extract
For pptn lead acetate
of tannins
 lead actate sol
For pptn of pass H2S
Lead sulphide
Extract
filtered

Crude Glycosides
Purification

 Fractional solubility
 Fractional crystallization
 TLC & column chromatography

III) VOLATILE OILS i.e. TERPENES & TERPENOIDS:

Also called as essential oil refers characteristic scent that develops the exalted
feeling after in inhaling the fragrance.
(A) Hydrodistillation - 4 methods of isolation are:

Method - 1 :Water distillation

Plant material
at the bottom of container
 Immersed in water
closed boil
vessel
Extract
(complete isolation not occur)

Method -2 : Water & steam distillation

Plant material
lodged on perforated grid or screen

Incontact with steam

Decomposition of oil less

yield of volatile oil more.

Method ;- 3 : Steam distillation

Plant material
Peoforated supported on grid
coils placed below
grid
Oils/Perfumes

* Condition
1. High pressure : Volatile oil likes to decompose (hydrolysis of ester)
2. Reduced presssure : Oils containing ester
3. Superheated steam distillation: Chemically stable compound.

Method - 4: Distillation per Se (terpentine)

Plant material
Org solvent/alcohol

Resin remain is molten form and

Support separation of less volatile fraction
Extraction of volatile oils is mainly done by means of "Clevenger apparatus.

DETERMINATION OF PERCENTAGE OF VOLATILE OIL IN DRUGS

Setting of the apparatus:

Apparatus for the determination of volatile oil in drugs consist of

(i) A round- bottom 1 litre boiling flask
(ii) A special still head.
Still head contains:
(i) Condenser (ii) receiver (iii) return tube.
A side tube is also attached to introduce water in the graduated tube and return tube
The official method for the estimation of volatile oil in a crude drug is hydro-
distillation based on distilling the drug with water and distillate is collected in the
graduated tube, from which the aqueous portion of the distillate automatically returns to
distillation flask.
Better results are obtained with some drugs by using 150 ml each of glycerine and
water in place of pure after in the boiling flask. This modification should be used for dill,
coriander, cloves, fennel and dried orange peel.
When the oil is heavier than water (e.g. clove oil) a known volume of xylene is
added to the graduated tube by a pipette which is passed through the side-tube of the still
head. The increase in volume of xylene gives volume of oil distilled.

Procedure:
(i) Take 10 to 20g of powdered drug with 250 to 300 ml of water in distillation flask.
Add a few pieces of porcelain to it (to avoid bumping during distillation)
(ii) Set up the apparatus as represented in the figure.
(iii) Fill the tubes (receiver and return tube) with water by introducing it at side tube
by means of a pipette. Close the side tube.
(iv) For heating the flask - Bunsen burner and asbestos filled wire gauge or heating
mantle can be used.
(v) Lift the flask at intervals and shake the contents, until the liquid is boiling
steadily. Finally, adjust the flame so that the distillate in the graduated tube remains cold.
Continue heating till no more oil collects . (This requires two hours or more).
(vi) Turn out the gas and allow the liquid in the condenser to drain for five or ten
minutes then read the volume of oil.
Express the result as a volume in weight percentage.

Diagram
(B) Extraction with non-volatile solvents i.e. fat

(1) With out of heat (Enfluerage or absorption)

used for extraction of delicate perfumes

In wooden frame (chassis) supported with glass plate in the middle

Spread fat layer of 3mm thickness (on either side of glass)

 Spread layer of flower (petals) over fat layer

Spread a fat layer over flower petals

such 35-50 chassis filed over one another

Flowers enclosed bet two layers of fats, release their aromatic components to
fats.

Length of exposure depends on flowers

(Jasmine - 24 hours, Jonquil - 48 hours & Tuberose - 72 hours)

After requiste time flowers are removed (fingers) or spatula

Process repeated until suff. oil absorbed over fat

Pomade (Product)
with strong alcohol

Oils & least traces of fats by freezing

* Commonly used fat contains

40 parts of beef fat & 60 parts of lard.

Least traces of fats by freezing

Residence (product) kept in freezer (to form ice) & immediately comes back it into room
temp, so that water content or solvent may melt away.
This process in continue until we get crude from of fat left behind.

2) With aid of heat (Maceration)

Fats used like (beef fat, lard, olive oil or paraffin oil)

Flowers (Petals) immersed in fats

(depend upto plant material)
for 24-48 hours 50 - 700 C

used hydraulic presses or centrifuges

 Express fat

Partly aromatised fat treated with fresh batches 10-15 times (until desired strength as to
odour)

Pomade (Product)
Steam distillat (similar of vol oils)

Oils
* Used for reparation of oil from Roses Oranges blossoms, Lilies of the valley & violets.

(C) Eculle
Used for extract of citrus oils
Oil Cells or seeds in rind

Ruptured mechanically using pointed projections

twisting raw material over them

clockwise direction

Product i.e. oil

(IV) TANNINS:
Organic, non-nitrogenous plant product have astringent properties.
Plant material
For tannic acid H2O/alcohol

alcoholic extract
ether

Etheral layer aqueous layer

 (Gallic acid & (True tannins)
ellagic acid)

Concentrated Concentrated

Subjected to isolation & purification by chromatography

(V) RESINS
Complex amorphous product which on heating first gets softened & then melts.
Insoluble in most polar & non polar solvent, but dissolves in alcholed, rolvent ether,
benzene & CHCl3
Crude drug
alcohol
alcoholic extract
Add large Proportion concentrated
H2O
ppt of Resin
Purification

Resins

Alkaloid:
Definition: the term “alkaloid” (alkali-like) is commonly used to
designate basic heterocyclic nitrogenous compounds of plant
origin that are physiologically active.

Deviation from Definition:

  Basicity: Some alkaloids are not basic. e.g.
Colchicine,Piperine, Quaternary alkaloids.
 Nitrogen: The nitrogen in some alkaloids is not in a
heterocyclic ring e.g. Ephedrine, Colchicine, and Mescaline.
 Plant Origine: Some alkaloids are derived from Bacteria,
Fungi, Insects, Frogs, and Animals.

Or,
According to Landenberg "Alkaloids are defined as natural plant
compounds that have a basic character and contain at least one nitrogen
atom in a heterocyclic ring and having biological activities."

Or,
According to characteristic features “Alkaloids are basic nitrogenous
plant origin, mostly optically active & possessing nitrogen hetero cycles as
there structural units with physiological action.”

Distribution and occurrence:

 Rare in lower plants.

 Dicots are richer in alkaloids than Monocots.
 Families rich in Alkaloids: Apocynaceae, Rubiaceae, Solanaceae and
Papaveracea.
 Families free from Alkaloids: Rosaceae, Labiatae

Distribution in Plant:

• All Parts e.g. Datura.

• Barks e.g. Cinchona
• Seeds e.g. Nux vomica
• Roots e.g. Aconite
• Fruits e.g. Black pepper
• Leaves e.g. Tobacco
• Latex e.g. Opium

Forms of Alkaloids:
  Free bases
 Salts with Organic acids e.g. Oxalic, acetic acids
 Salts with inorganic acids e.g. HCl, H2SO4.
 Salts with special acids: e.g. Meconic acid in Opium,
Quinic acid in Cinchona.
 Glycosidal form e.g. Solanine in Solanum.

Function in Plants:

 They may act as protective against insects and herbivores due to their
bitterness and toxicity.
 They are, in certain cases, the final products of detoxification (waste
products).
 Source of nitrogen in case of nitrogen deficiency.
 They, sometimes, act as growth regulators in certain metabolic
systems.
 They may be utilized as a source of energy in case of deficiency in
carbon dioxide assimilation.

Properties of Alkaloids
Physical Properties:
 Physical form or Nature:
• Most alkaloids are crystalline solids.
• Few alkaloids are amorphous solids e.g. emetine.
• Some are liquids that are either:
 Volatile e.g. nicotine and coniine, or
 Non-volatile e.g. pilocarpine and hyoscine.
 Color:
• The majority of alkaloids are colorless but some are colored
e.g.:Colchicine and berberine are yellow.
• Canadine is orange.
• The salts of sanguinarine are copper-red.
Solubility:

• Both alkaloidal bases and their salts are soluble in alcohol.

• Generally, the bases are soluble in organic solvents and insoluble in
water
 Exceptions:
 • Bases soluble in water: caffeine, ephedrine, codeine,
colchicines, pilocarpine and quaternary ammonium
bases.
• Bases insoluble or sparingly soluble in certain organic
solvents: morphine in ether, theobromine and
theophylline in benzene.

Isomerization:
• Optically active isomers may show different physiological activities.
 L-ephedrine is 3.5 times more active than d-ephedrine.
 L-ergotamine is 3-4 times more active than d-ergotamine.
 D- Tubocurarine is more active than the corresponding L- form.
 Quinine (l-form) is antimalarial and its d- isomer quinidine is
antiarrythmic.
 The racemic (optically inactive) dl-atropine is physiologically
active.

Chemical Properties: Alkaloid is basic in nature because of lone pair of

electron on nitrogen atom.

 Nitrogen:
• Primary amines R-NH2 e.g. Nor ephedrine
• Secondary amines R2-NH e.g. Ephedrine
• Tertiary amines R3-N e.g. Atropine
• Quaternary ammonium salts R4-N e.g. d-Tubocurarine

 Basicity:
• R2-NH > R-NH2 > R3-N
• Saturated hexacyclic amines are more basic than aromatic
amines.
According to Basicity Alkaloids are classified into:
• Weak bases e.g. Caffeine
• Strong bases e.g. Atropine
• Amphoteric e.g. Morphine
• Neutral alkaloids e.g. Colchicines
 Oxygen:
• Most alkaloids contain Oxygen and are solid in nature e.g.
Atropine.
 • Some alkaloids are free from Oxygen and are mostly liquids
e.g. Nicotine, Coniine.
 Stability:
• Effect of heat: Alkaloids are decomposed by heat, except
Strychnine and caffeine (sublimable).
• Reaction with acids:
 Salt formation.
 2- Dilute acids hydrolyze Ester Alkaloids e.g. Atropine
• Conc. acids may cause:
Dehydration:
Atropine → Apoatropine
Morphine → Apomorphine
• Effect of Alkalies:
 Dilute alkalis liberate most alkaloids from their salts e.g.
NH3.
 They may cause Isomerization (racemization) of
alkaloid as the conversion of Hyoscyamine to atropine.
 Strong alkalis: such as aqueous NaOH and KOH form
salts with phenolic alkaloids.
 Strong alkalis cause hydrolysis of Ester alkaloids
(e.g. atropine, cocaine and physostigmine) and Amide
alkaloids (colchicines).
 Strong alkalis cause opening of lactones ring.