Phytochemical analysis of the plant

The plant material
Ideally, fresh plant tissues should be used for phytochemical analysis and the material should be plunged into boiling alcohol within minutes of its collection.
Alternatively, dried plants may be used for extraction but it is essential that drying operation is carried out under controlled condition. Extraction of flavonoids, alkaloids, quinones and terpenoids can be carried out successfully using dried plant material.

Extraction
The goal of any extraction procedure is to isolate the plant's active principles. To achieve this goal several extraction procedures may be considered depending not only on the type of major phytochemical groups such as alkaloids, glycosides, flavonones, organic acids etc. but also on solubilities in water and various solvent combinations, molecular weights, polarity and degree of ionizability.
Alcohol is a good solvent for preliminary extraction and to prevent enzymatic oxidation or hydrolysis fresh leaf or flower tissues are plunged into boiling alcohol. The following are some examples of isolation of active principles –

Alkaloids
First of all the presence of an alkaloid is ascertained by treating plant extract with various alkaloidal reagents such as potassium mercuric iodide (Mayer's reagent), iodine dissolved in potassium iodide (Wagner's reagent), potassium bismuth iodide (Dragendroff's reagent), tannic acid, picric acid, phosphomolybolic acid (Sonneuschein's reagent) and phosphotungstic acid (Scheibler's reagent) with which alkaloids give characteristic precipitate or turbidity. A common method for the isolation of alkaloid as recommended by Manske is as follows –
The dried and powdered plant material is first extracted with petroleum ether (if it is rich in fat) and then filtered for the removal of soluble fats. The residue is then extracted with methyl alcohol to remove cellulose and other insoluble material and the filtrate so obtained is evaporated. This is usually carried out in a rotary evaporator, which will concentrate bulky solutions down to small volumes without bumping. The evaporated mass is dissolved in water, acidified to pH 2 and finally steam distilled to remove methyl alcohol. The dark residual solution is allowed to stand for several days in a refrigerator to remove suspended impurities. The filtrate is extracted with ether or chloroform to remove water soluble non basic organic material and then steam distilled when the steam volatile alkaloids are separated. The solution of the rest of the alkaloid salts is made alkaline and again extracted with ether or chloroform. The ether layer obtained after this extraction is evaporated to give crude alkaloids. The whole process is graphically depicted as below :-

Alternative Procedure
Alkaloids may be extracted from plants into a weakly acid (1m HCl or 10% acetic acid) alcohol solvent and then precipitating with concentrated ammonia.
Extract dried tissue with 10% acetic acid in ethanol, leaving to stand for at least hour. Concentrate the extract to one quarter of the original volume and precipitate the alkaloid by dropwise addition of conc. NH4OH. Collect by centrifugation, after washing with 1% NH4OH. Dissolve residue in few drops of ethanol or chloroform.
Chromatograph an aliquot on silica gel G plates in methanol conc. NH4OH (200:3). Detect the presence of alkaloids on the plate, first of all by any fluorescence in UV light and then by spraying Dragendorff, Iodoplatinate and Marquis reagent -
Dragendorff's reagent
Solution A - 0.6 g Bismuth subnitrate in 2 ml conc. HCl and 10 ml water
Solution B - 6 g potassium iodide in 10 ml water.
Mix solution A & B together with 7 ml conc. HCl and 15 ml water and then dilute with 400 ml water. After spray it will give orange brown spots on a yellow background.

Iodoplatinate reagent
10 ml of 5% platinum chloride, 5 ml conc. HCl and 240 ml 2% potassium iodide are mixed. On spray it gives different colours.

Marquis reagent
1 ml of formaldehyde in 10 ml conc. H2SO4. It gives yellow to purple spots.

Flavonoids
Flavonoids can be extracted from the plant tissue by following procedure –

• A small amount of plant tissue (leaf or flower) after immersing in 2 m HCl is heated in a test tube for 30-40 min at 100oC.
• The cooled extract is then filtered and extracted with ethylacetate.
• If the solution is coloured then the aqueous extract is again heated to remove last traces of ethyl acetate and reextracted with a small volume of amyl alcohol.
• The ethyl acetate is concentrated to dryness, taken up in 1-2 drops of ethanol and aliquot is chromatographed in following solvents. Forestal acetic acid conc. HCl water : 30:3:10, 50% aqueous acetic acid, Butanol : acetic acid : water : 4:1:5.
• The amyl alcohol extract, which should be coloured, is concentrated to dryness, taken up in a few drops of 1% methanolic HCl and aliquot chromatographed in Forestal and in formic acid : conc HCl : Water : 5:2:3.