Pharmacognosy & Phytochemistry - II

Unit III

ISOLATION, IDENTIFICATION AND ANALYSIS OF PHYTOCONSTITUENTS

Points to be covered.

1. Terpenoids : Menthol, Citral, Artemisin

2. Glycosides: Glycyrrhetinic acid and Rutin

3. Alkaloids : Atropine, Quinine, Reserpine, Caffeine

4. Resins : Podophyllotoxin, Curcumin.

TERPENOIDS:

1. MENTHOL:

Synonym: Menthol

Biological source: Menthol is a monoterpene alcohol obtained from oils of Mentha piperita var. Vulgaris (Black peppermint) and Mentha piperita var. officinalis (White peppermint), belongs to family - Labiatae.

Isolation:

Menthol is isolated by two Method

1: Hydro-distillation Method

2: Steam distillation

1. Hydro-distillation method:

i. Required quantity of coarse powder leaves of Mentha piperita

ii. The peppermint oil is extract by hydrodistillation method by using

Clevenger apparatus

iii. The oil is separated from water and allow to cooling

iv. After cooling crystals of (-) menthol will separate out

v. The crystals are collected by centrifugation and re- crystallized the

menthol by acetone or any other low boiling point solvent.

2. Steam distillation:

i. Required quantity of air dried mentha plant is charged into stainless steel still having perforated bottom.

ii. The steam under pressure is generated with the help of boiler and steam is passed through the drug

iii. The mixture vapour (water and volatile oil) are passed through the condenser where vapour is cooled and back to liquid form

iv. The mentha oil is collected in separating can

v. Mentha oil is floated on top of the water due to lighter than water

vi. The oil is then decanted and filtered

vii. The oil is allowed to cool; crystal of menthol will separate out.

Properties:

Appearance: White crystalline substances, which is solid at room temperature and melts slightly above (m.p. 41 to 43°c).

Odour : Characteristic and pleasant.

Taste: Pungent followed by cooling sensation.

Solubility : Soluble in 70% alcohol, ether and chloroform, insoluble in water.

Identification by chemical test:

Few drops of sample is mixed with 5ml of nitric acid and heated on water bath Blue colour is developed within 5 minutes, after some time it becomes yellow colour Indicate the presence of menthol

Analysis by TLC:

Sample preparation : 1mg of Menthol is dissolved 1ml of methanol

Standard sample : Menthol

Stationary phase: Silica gel -G

Mobile phase: Pure Chloroform

Detecting agent :1% vanillin - sulphuric acid reagent and heat the plate at 110°C for 10 minutes

RF Value: 0.48-0.62.

2. CITRAL::

Synonym: Citral

Biological source: Citral is a monoterpene aldehyde obtained from oils of Cymbopogon flexuosus, C.

martini belongs to family- Graminae.

It contains not less than 75% of aldehyde calculated as Citral. It also present in Lemon oil (Citrus limonus) and Orange oil (Citrus aurantium), family Rutaceae.

Isolation:

Citral is isolated by following methods

1. Hydro-distillation

2. Fractional crystallization Method

In case of lemon grass, the fresh plant material is hydrodistilled to obtain lemon grass oil
In case of citrus fruits, the oil is separated either by eculle process or by expression method.
The oil, if obtained from citral fruits, is not kept in contact with water for long as the white albino or zest degrades the oil
Citral has a low boiling point, therefore it is not hydrodistilled due to the risk of degradation.
The fractional distillation of oil gives a low boiling component containing higher citral content
Fractional crystallization isa suitable method for the purification of Citral

3. Fractional crystallization:

First sodium sulphite is added to the total oil
Thus the Citral gets converted to its sulphite salt
The salt crystallizes out of the solution
The crystals are filtered and washed with ether or chloroform
The product is then subjected to sodium carbonate treatment to recover Citral
Citral is found in two form i.e Citral-a (Geranial) and Citral-ß (Neral)

Properties:

Appearance : Clear pale yellow liquid

Odour: Strong lemon like odour

Taste : Lemon like taste

Solubility : Soluble in 3 parts of 70% alcohol, chloroform and fixed oil. Insoluble in water

Boiling point: 224-228°C.

ldentification by chemical test:

1. Alcoholic solution of Sudan red IIl is added to the sample Red colour is appeared which indicate presence of citral

2. Tincture alkane is added to the sample. Red colour is appeared which indicate presence of Citral

Analysis by TLC Sample preparation:

Sample preparation : 1mg of Citral is dissolved 1ml of methanol

Standard sample : Citral

Stationary phase : Silica gel -G

Mobile phase: Pure Chloroform

Detecting agent : 2, 4, dinitrophenyl hydrazine reagent

Color spots : Yellow to orange

RF Value: 0.51.

ARTEMISIN:

Synonym: Artemisin, Santonica, worm seed

Biological source: Artemisin is a sesquiterpenoid lactone, obtained from the unexpanded flower- heads of, Artemisia brevifolia Artemisia maritime and other species belongs to family : Asteraceae.

It contains 2-3% of essential oil

and two crystalline substances i: e Santonin and Artemisin.

Isolation:

i. The air dried powdered of herb is macerated in methanol for 1 hour with magnetic stirrer having speed of 700 rpm

i. This process is repeated until methanol layer become colorless

ii. The extract is evaporated by rota-evaporator at a temnperature of 40°C until volume is reduced to 100ml

iv. The methanolic extract is partitioned using 50ml hexane (Hexane: Methanol, 1:2) until colorless hexane layer is obtained

V. Hexane and methanol extract is separated using funnel

vi. 10ml of distilled water is added to methanolic extract and partitioned by 50ml of ethyl acetate until ethyl acetate layer become colorless

vi. Again ethyl acetate and methanolic extract is separated

viii. Both extracts are concentrated by using rota evaporator at a temperature of 40°C

ix. Artemisin is fractionated by column chromatography using silica gel 60 as stationary phase and ethyl acetate: hexane as mobile phase.