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Isolation,identification and analysis of phytoconstituents. Dr.U.Srinivasa, Professor and Head, Srinivas college of Pharmacy, Mangalore-Karnataka

This document provides information on the isolation and analysis of several compounds: 1. Quinine is isolated from cinchona bark through extraction with benzene followed by crystallization. It is analyzed using thin layer chromatography. 2. Glycyrrhetinic acid is the active compound in liquorice root. It is isolated through chloroform extraction and acid hydrolysis, and analyzed with chemical tests and thin layer chromatography. 3. Rutin is isolated from eucalyptus leaves by boiling in water and recrystallization. It is analyzed using chemical tests and thin layer chromatography. 4. Podophyllotoxin, the active compound in mayapple, is extracted with methanol and benzene from

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QUININE Synonym – Quinine It is a quinoline alkaloid of cinchona bark. The other important alkaloids of this drug are quinidine, cinchonine, cinchonidine, cinchonamine etc., Biological sources : It consists of dried inner bark of C.Calisaya, C.succirubra, C.officinalis, C.ledgeriana and hybrids of this. Family – Rubiaceae
Quinine and quinidine are stereo-isomers . Quinine is levorotatory and quinidine is dextrorotatory Uses : Quinine is antimalarial Quinidine is a cardiac depressant therefore used in cardiac arrhythmias.
ISOLATION 1. The dry powder bark material is first well mixed with about 30% of its weight of calcium hydroxide or calcium oxide and sufficient quantity of sodium hydroxide solution to make a paste. It is allowed to stand for few hours. 2. The mass is then transferred to a Soxhlet apparatus and extraction is carried out with benzene.
3.Subsequently the benzene extract is shaken with successive portions of 5% sulphuric acid. 4.The aqueous acid extract is adjusted the pH 6.5 with dilute sodium hydroxide, cool. Crystals of neutral quinine sulphate are formed. 5.These crystals are freed from cinchonine and cinchonidine by repeated recrystallization from hot water.
6.Colouring matter is removed by activated charcoal. 7.Quinine sulphate crystals are dissolved in dilute sulphuric acid and made alkaline with ammonia. Initially amorphous quinine is formed , which becomes crystalline. 8.Finally washed to remove sodium and ammonium salts and dried to 45- 55 o C.
IDENTIFICATION AND ANALYSIS 1. Chemical tests 2. Thin layer chromatography (TLC)
T.L.C Method Sample preparation – Dissolved about 1mg of Quinine or Cinchona alkaloid in 1ml of methanol Stationary phase - Silica gel –G Detecting agent – Dragendroffs reagent Mobile phase – Chloroform – Diethylamine (9:1) RF Value – Quinine – 0.17, Quinidine -0.26
GLYCYRRHETINIC ACID Biological source - It is obtained from the roots and subterranean stems of Glycyrrhiza glabra Family – Leguminosae Chemical Constituents – A major component is sweet triterpenic saponin glycoside , glycyrrhizin Glycyrrhizin – It is a potassium and calcium salt of Glycyrrhizic acid Glycyrrhetinic acid is a Pentacyclic triterpenoid aglycone. It is used as an antiulcer.
ISOLATION • The Liquorice / Glycyrrhiza coarse powder is extracted with chloroform. • Filter and discard the filtrate. • Extract the marc with 0.5 M Sulphuric acid for a few hours
• Filter and extract the filtrate with three portions of chloroform • Separate and combine the chloroform layers • Distill off the chloroform extract to yield a dry residue of glycyrrhetinic acid. • White crystalline powder, insoluble in water, soluble in chloroform, benzene, ether etc
IDENTIFICATION AND ANALYSIS 1. Chemical tests – Liebermann test and Liebermann – Burchard test 2. Thin layer chromatography (TLC)
T.L.C Method Sample preparation – Dissolved about 1mg of Glycyrrhetinic acid in 1ml of methanol- Chloroform (1:1) Stationary phase - Silica gel –G Detecting agent – 1% vanillin- Sulphuric acid and heat for 10 minutes at 1100 C Mobile phase – Toluene–Ethyl acetate-Glacial acetic acid (12.5:7.5:0.5) Reference drug - Glycyrrhetinic acid RF Value – Purplish – 0.41
RUTIN • There are around 200 types of Quercetin, Flavanoid glycosides, among this the rutin is the one of most important type. • It is chemically Quercetin-3- rutinoside . On hydrolysis , it yields the aglycone quercetin and the sugars glucose and rhamnose. • It is used as a Vitamin P OR Capillary fragility factor
SOURCES OF RUTIN • 1. Fagophyrum esculentum- ( Polygonaceae ) • 2. Rhubarb – ( Rheum emodi- ( Polygonaceae ) • 3. Tobacco – (Nicotiana tobaccum –( Solanaceae ) • 4. Ruta – (Ruta graveolens – ( Rutaceae ) • 5. Tea – Thea sinensis – ( Theaceae ) • 6. Eucalyptus macroryncha ( Myrataceae )
ISOLATION • Source - • Eucalyptus macroryncha ( Myrataceae ) • Boil the powder drug with boiling water.Filter while hot and collect the filtrate . Cool for the precipitation of the rutin. • Recrystallize it from boiling water , dry the product. • Greenish yellow crystalline powder
IDENTIFICATION AND ANALYSIS 1. Chemical tests – Shinoda test 2. Thin layer chromatography (TLC)
T.L.C Method Sample preparation – Dissolved about 1mg of Rutin in 1ml of methanol Stationary phase - Silica gel –G Mobile phase – 10 % aqueous sodium chloride solution Standard drug - Rutin RF Value – Yellow spot – 0.43
PODOPHYLLOTOXIN • Podophyllotoxin is the Lactone resin present in the root and rhizome of Podophyllum hexandrum • Family – Berberidaceae • It is used as anti-proliferative agent (Anti cancer agent)
EXTRACTION • Take the weighed quantity of rhizomes or roots of Podophyllum emodi with methanol. Filter and evaporate to semisolid mass. • Dissolve semisolid mass into acidic water. Precipitate is formed which should be allowed for at least for 2hrs
• Filter and wash filtrate with cold water. Collect the residue, wash with acidified water and dry to obtain dark brown amorphous powder. • Extract the residue with hot alcohol. Filter and evaporate to dryness. • Re- crystallise the residue in benzene to yield podophyllotoxin
IDENTIFICATION AND ANALYSIS • 1. Chemical test – Treat podophyllotoxin with 50% Sulphuric acid it will show violet – blue colour. • 2.Thin layer chromatography ( TLC )
T.L.C Method Sample preparation – Dissolved about 1mg of podophyllotoxin in 1ml of methanol Stationary phase - Silica gel –G Mobile phase – Chloroform : Methanol (90:10) for about 6 cm (Only glycosides are separated but aglycone like podophyllotoxin remains in the region of the front.
The same plate is again eluted with more weakly polar Solvent Chloroform : Acetone (65:35) upto 12cm Standard drug – Podophyllotoxin Detection – Spray with methanol Sulphuric acid and heat 10 minutes at 1100 C RF Value – Yellow spot – 0.65