Isolation, Identification and Analysis of Phytoconstituents

Isolation, Identification and Analysis of Phytoconstituents
a) Terpenoids: Menthol, Citral, Artemisin
b) Glycosides: Glycyrhetinic acid & Rutin
c) Alkaloids: Atropine,Quinine,Reserpine,Caffeine
d) Resins: Podophyllotoxin, Curcumin

1. Isolation, Identification and Analysis of Phytoconstituents DR. SIDDHI UPADHYAY H.O.D. & ASSOCIATE PROFESSOR Dept. of Pharmacognosy and Phytochemistry SIGMA INSTITUTE OF PHARMACY

2. CONTENT Terpenoids: Menthol Citral Artemisin Glycosides: Glycyrhetinic acid Rutin Alkaloids: Atropine Quinine Reserpine Caffeine Resins: Podophyllotoxin Curcumin

3. 🠶 B i o l o g ic a l so u rce – 🠶 Menthol is a monoterpene alcohol obtained from different variety of mint oils or peppermint oils 🠶 Biological source – It consists of the fresh flowering tops of Mentha piperita, Mentha officinalis 🠶 Fami l y - La b i a tat e 🠶 Granular substance or crystalline with peppermint taste and odour, freely soluble in alcohol, chloroform, ether, petroleum ether MENTHOL

4. EXTRACTION AND ISOLATION 🠶 Take the accurately weighed quantity of coarse powder of Mentha piperita parts just before flowering . 🠶 Extract the peppermint oil by water distillation method. 🠶 Separate the oil and allow cooling. Crystals of (-) menthol will separate out. 🠶 C o llect the cr y stals by ce n tr i fug a tion. 🠶 Re- Crystallize menthol from acetone or any other low boiling point solvent

5. Identification and Analysis T.L.C Method Sample preparation – Dissolved 1mg of menthol in 1ml of methanol Stationary phase - Silica gel –G Standard sample - Menthol Detecting agent – 1% vanillin – sulphuric acid reagent and heat the plate 110 C for 10 minutes Mobile phase – Pure Chloroform Rf Value – 0.48-0.62

6. 🠶 Biolo g ic a l s o urc e s – 🠶 Citral is a monoterpene aldehyde found in variety of sources like lemon grass, lemon and orange peels etc 🠶 Cymbopogon fleuosus ( Lemon grass) , Graminae . 75-85% of citral present in the drug 🠶 Citral o b tained from a natural s o urce is a mixture of two geometric isomers geranial and neral CITRAL

7. 🠶 Pro p ert i es 🠶 Gera n ial a n d N e ral b o th are light oily liquids with lemon odour 🠶 Citral is practically insoluble in water but miscible with alcohol, ether, benzyl benzoate etc

8. EXTRACTION AND ISOLATION 🠶 T h e fre s h p la n t m a terial is h y dr o - di s ti l led to obtain lemon grass oil. 🠶 Purification by Fractional crystallization 🠶 To the total oil, first Sodium sulphite is added, the citral get converted into its sulphite salt 🠶 T h e s alt cr ys talliz e s o u t o f the s ol u tion 🠶 The crystals are filtered and washed with ether or chloroform 🠶 The product is then subjected to sodium carbonate treatment to recover citral

9. Identification and Analysis T.L.C Method Sample preparation – Dissolved 1mg of Citral in 1ml of methanol Stationary phase - Silica gel –G Standard sample - Citral Detecting agent – 2,4,dinitrophenyl hydrazine reagent to produce Yellow to orange Color spots Mobile phase – Pure Chloroform RF Value – 0.51

10. 🠶 S y n o n y m – Sa n toni c a 🠶 Biological source – Artemisin is a sesquiterpenoid lactone , obtained from the unexpanded flower- heads of Artemisia annua 🠶 F a mily - C o m p o s itae 🠶 Medi c inal use – Antimalarial drug 🠶 White crystalline powder, soluble in organic solvents ARTEMISIN

11. EXTRACTION AND ISOLATION 🠶 The leaves are air dried, coarsely powdered and extracted with petroleum ether (40-60). 🠶 The extract is concentrated ,dried and re-dissolved in chloroform. Add acetonitrile to precipitate sugars and waxes. 🠶 Filter and collect the filtrate. Evaporate to dryness to yield residue

12. 🠶 The chromatographic fractionation of the concentrate on silica gel by eluting with Chloroform- ethyl acetate yields the fraction of artemisin 🠶 The fractions containing artemesin could be crystallised from cyclohexane or 50% Ethanol

13. Identification and Analysis T.L.C Method Sample preparation – Dissolved 1mg of Artemisin in 1ml of Chloroform Stationary phase - Silica gel –G Standard sample - Artemisin Detecting agent – p- dimethylaminobenzaldehyde and heat at 80  C to produce color Mobile phase – Petroleum ether - Ethyl acetate (1:2) RF Value – Compare with standard Artemisin

14. 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. GLYCYRHETINIC ACID

15. 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

16. 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

17. IDENTIFICATION AND ANALYSIS Chemical tests – Liebermann test and Liebermann – Burchard test Thin layer chromatography (TLC)

18. 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 110 C Mobile phase – Toluene–Ethyl acetate-Glacial acetic acid (12.5:7.5:0.5) Reference drug - Glycyrrhetinic acid RF Value – Purplish – 0.41

19. 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 RUTIN

20. 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 )

21. 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

22. IDENTIFICATION AND ANALYSIS Chemical tests – Shinoda test Thin layer chromatography (TLC)

23. 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

24. 🠶 Atropine i s a trop a ne al k alo i d o b tained from A t ropa belladonna ,Datura stramonium and Hyoscyamus niger 🠶 Family – Solan a ce a e 🠶 U s ed as Antis p a s mo d ic, M y driatic etc ATROPINE

25. EXTRACTION AND ISOLATION 🠶 Take weighed quantity of coarse powder and moisten with sodium carbonate solution. 🠶 Extract the blended mixture in petroleum ether. Filter the petroleum ether extract 🠶 E x tract the fi l trate with aqueous a c et i c a c id ( al k alo i ds extracted in aqueous layer) 🠶 Extract the aq u eous f r acti o n with sol v ent ether and separate both fraction. Discard solvent ether fraction

26. Identification and Analysis 🠶 C h emi c al test – 🠶 V ital i n –morin test - 🠶 Take small quantity of the solid atropine and add 2 drops of Con.nitric acid in an evaporating dish and evaporated to dryness on water bath. Then dissolve the residue in 1ml of acetone. Add few drops of freshly prepared alcoholic potassium hydroxide solution. 🠶 Violet colouration takes place due to tropane nucleus

27. T.L.C Method Sample preparation – Dissolved 1mg of Atropine in 1ml of Chloroform Stationary phase - Silica gel –G Standard sample - Atropine Detecting agent – Drangendroffs reagent to produce yellow orange Color spots Mobile phase – Toluene - Ethyl acetate – Diethyl amine (70:20:10) RF Value – Compare with standard Atropine (0.70)

28. 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

29. 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.

30. ISOLATION 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. The mass is then transferred to a Soxhlet apparatus and extraction is carried out with benzene.

31. Subsequently the benzene extract is shaken with successive portions of 5% sulphuric acid. The aqueous acid extract is adjusted the pH 6.5 with dilute sodium hydroxide, cool. Crystals of neutral quinine sulphate are formed. These crystals are freed from cinchonine and cinchonidine by repeated recrystallization from hot water.

32. Colouring matter is removed by activated charcoal. Quinine sulphate crystals are dissolved in dilute sulphuric acid and made alkaline with ammonia. Initially amorphous quinine is formed , which becomes crystalline. Finally washed to remove sodium and ammonium salts and dried to 45- 55 o C.

33. IDENTIFICATION AND ANALYSIS Chemical tests Thin layer chromatography (TLC)

34. 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

35. 🠶 Biolo g i c a l s o u rce – R e serp i ne i s an ind o le al k aloid obtained from the roots of Rauwolfia serpentina 🠶 F a mily – Ap o c y a n a c e a e 🠶 I t i s a w h ite or p al e b u f f t o slig h t l y yello w , o d our l ess, crystalline powder 🠶 It is soluble in alcohol, acetone and chloroform. 🠶 Reserpi n e i s an a ntih y p e rt e n s i ve and a n tip s ych o tic agent RESERPINE

36. EXTRACTION AND ISOLATION 🠶 R a u w o lf i a root p o wder i s e x haustiv e l y extra c ted with 90% alcohol by percolation 🠶 The alcoholic extract is concentrated and dried under reduced pressure below 60  c to yield Rauwolfia dry extract. 🠶 Rauwolfia dry extract is extracted with Ether-chloroform- 90%alcohol (20:8:2.5) 🠶 Collect the extract and add little dilute ammonia with intermittent shaking. Add water and allow the drug to settle after vigorous shaking.

37. Identification and Analysis T.L.C Method Sample preparation – Dissolved 1mg of Reserpine in 1ml of methanol or chloroform Standard sample - Reserpine Stationary phase - Silica gel – G Mobile phase – Chloroform: acetone :diethyl amine (50:40:10) Detecting agent – Dragendroffs reagent RF Value – 0.72

38. 🠶 Ca f fei n e i s a p u ri n e alkal o id o b tai n ed f rom T e a leav e s, Coffee seeds, cocoa, and other species 🠶 Biological source -It consists of dried leaves of plant known as Thea sinensis 🠶 Family – The a ce a e 🠶 It i s ch e mic a lly 1,3,7, t rim e thyl x a n thi n e . It i s isolat e d fr o m tea and coffee seeds during decaffeination process. 🠶 Tea leaves contains 1-4% of caffeine and coffee contains 1- 2% of caffeine 🠶 It i s white p o w d er or white ,glist e ring needles, o dour less, bitter in taste, Soluble in hot water. 🠶 Caffeine is a CNS stimulant and Diuretic CAFFEINE

39. EXTRACTION AND ISOLATION 🠶 Th e powder tea lea v e s i s e xtra c ted wi t h b o iling w a ter and the aqueous extract is filtered while hot. 🠶 Th e wa r m e x tr a ct i s trea t ed with lead a c e t ate to precipitate tannins and filtered. 🠶 Th e fi l trate i s treated with e xcess o f dilute s u l p h uric acid to precipitate lead in the form of lead sulphate.

40. 🠶 Filter and c o llect the fi l trate 🠶 Th e fi l trate i s b o il e d with A c t iv a ted charc o al t o remove colouring matter, if any and filtered to remove charcoal 🠶 Th e fi l tered d e c o l o u rized solution i s e x tract e d with chloroform successively . 🠶 Combin e d the c h lo r oform e x t racts e vaporate on w a ter bath to yield caffeine (white powder) 🠶 I t i s re c ry s talliz e d with al c o h ol

41. Identification and Analysis 🠶 C h emi c al test – 🠶 Murexide test – To the caffeine add hydrochloric acid and potassium chlorate, heated to dryness. A purple colour is obtained by exposing the residue to vapours of dilute ammonia. 🠶 T h in la y er c h rom a togra p hy (TL C )

42. T.L.C Method Sample preparation – Dissolved 1mg of caffeine in 1ml of methanol or chloroform Stationary phase - Silica gel –G Standard sample - Caffeine Mobile phase – Ethyl acetate: methanol : acetic acid (80:10:10) Detecting agent – Expose to vapors of iodine RF Value – 0.41

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)

44. 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

45. 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

46. IDENTIFICATION AND ANALYSIS 1. Chemical test – Treat podophyllotoxin with 50% Sulphuric acid it will show violet – blue colour. 2.Thin layer chromatography ( TLC )

47. T.L.C Method Sample preparation – Dissolved about 1mg of podophyllotoxin in 1ml of methanol Stationary phase - Silica gel –G Mobile phase – C h loroform : M e tha n ol (90:1 ) f or a b o u t 6 cm (Only glycosides are separated but aglycone like podophyllotoxin remains in the region of the front.

48. 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 110 C RF Value – Yellow spot – 0.65

49. 🠶 C u r cumin or C u r cuminoids are the d iaryl h e p n oid compounds obtained from the dried rhizomes of Turmeric, Curcuma longa, Family – Zingiberaceae 🠶 C u r cumin i s the maj o r c o l o u ring pr i n c ipl e . I t i s a mixture of curcumin, monodesmethoxycurcumin and bisdesmethoxycurcumin CURCUMIN

50. 🠶 It as an ora n ge y e llo w , cr y stalline p o w d er 🠶 Insoluble in water and ether, but soluble in alcohol 🠶 It is used as wound healing, ant-inflammatory, anti arthritic and antimicrobial activities 🠶 Us e d ag a inst pe p tic ulcer

51. Identification and Analysis T.L.C Method Sample preparation – Dissolved 1mg of Curcumin in 1ml of methanol Stationary phase - Silica gel –G Standard sample - Curcumin Detecting agent – Observed under U.V light at 366nm Mobile phase – Chloroform - Ethanol - Glacial acetic acid (94:5:1) RF Value – Curcumin – 0.79

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