Extraction & isolation method of phyto constituents (individual)

(I) ALKALOIDSMeans '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 weakbases 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 aquendescrystallization Chromatographic fractionseperation etc.

Structure identification by modernanalytical 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 extractFlavonoids 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 limeAlkaloidal freebases(From their acid salts) CH Cl3/ether

Organic solvent layer aqueous layer(Concentrated) (Alkaloidal salt) Aqueous acid NH3 or

aq KOHAlkaloid Liberate freebase

Org. solvent

Organic extract(Concentrated)

Drying

Total alkaloid

Method - 4Manske'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 paraffinSeveral days (to remove impurities)(to remove impurities)

Filtrate Filtrate ether/CHCl3 ether/CHCl3

Remove non-alkaloidal To remove non alkaloidal impuritiesImpuritiesRemove 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 SolubleExtractedn- butanol

n -butanol extractconcetrated & pptwith acetone

ppt contains mixture of Glycoside

Method - 2:

Fresh plant material shake with n-butanol Shake with waterand ethylacetate (1:1)

Org-phase Aq. Phase

extract with evaporated extract withethylacetate invaccum n- butanol

Ethyl acetate Ethylacetate n-butanol (insoluble) extract

SiO2 column SiO2 column Glycoside Glycoside

Method - 3 : Stass Otto method

Powdered drugFor thermolabile Soxhlation with alcoholdrugs below 450 C Extract For pptn lead acetate

of tannins

lead actate sol For pptn of pass H2S

Lead sulphideExtract 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 waterclosed boilvessel 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

* Condition1. High pressure : Volatile oil likes to decompose (hydrolysis of ester)2. Reduced presssure : Oils containing ester3. 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 freezingResidence (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) EculleUsed for extract of citrus oilsOil 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 materialFor 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) RESINSComplex 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 extractAdd 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 AlkaloidsPhysical 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.