GYLCOSIDES

1. GYLCOSIDES Presented by BAHADUR SINGH HOD Department of Pharm. Sciences Govt. Polytechnic college for Girls Patiala

2. Glycoside Definition A substance which on hydrolysis • yields a reducing sugar • + one or more non-sugar substances (genin/aglycone) • Normally aromatic in nature • Known as the aromatic aglycone or genin portion

3. Glycoside = Broad & general termEmbraces all the many various combinations of sugars & aglyconese.g. Cardiac glycosides, flavonoidglycosides, phenolic glycosides etc

5. important to determine which isomer has the activityα or βglycosidal bond from an α or βpyranose sugar ringnatural glycosides tend to have β-linkageacid hydrolysis to cleave α or β glycosidesidentify component part of moleculecheck stereochemistry with β-glucosidase

6. Classification: • Atom from the aglycone involved in the glycosidic linkage: • Aglycone- O- Sugar O-glycosides • Aglycone- C- Sugar C-glycosides • Aglycone- S- Sugar S-glycosides • Aglycone- N- Sugar N-glycosides • Number of sugars: • One sugar monosidese.g. Salicin. • Two sugar Biosides e.g. Diosmin. • Three sugars Triosides e.g. Digoxin. • Nature of the glycoside: • Primary glycosides: Originally present in the plant e.g. Purpurea A • Secondary glycosides: Resulted from removal of one sugar from the primary glycosides e.g. Digitoxin

7. Type of the glycosidic linkage:a- glycosidesb- glycosidesBotanical source:Digitalis glycosidesSenna glycosides.Therapeutic use:Analgesic glycosides.Purgative glycosides.Cardiac glycosides.Chemical nature of the aglycone:Flavone glycosides.Steroidal glycosides.Aldehydic glycosides.MOST COMMONLY USED

8. Stability of Glycosides: 1- Effect of acid hydrolysis: • Acids split sugars from the aglycones. • The acetal linkage is more readily cleaved than the linkage between the individual sugars of the sugar chain. • C-glycosides are resistant to acid hydrolysis. 2- Effect of alkaline hydrolysis: A- Strong alkalis: • Hydrolysis of ester groups. • Opening of lactone rings e.g. Cardiac glycosides. B- Mild alkalis: • Hydrolysis of ester groups e.g. Lanatoside A to Purpurea A • Opening of lactone rings e.g. Cardiac glycosides

9. 3- Enzymatic hydrolysis:Split the sugars stepwise starting from the terminal sugars.All plants producing glycosides have enzyme that can hydrolyze these glycosides.Enzymes are specific for the type of glycosidic linkages:Emulsin can hydrolyze b- glycosidesInvertase can hydrolyze a- glycosidesMyrosin can hydrolyze s-glycosides.

10. Physical Characters: • Solids either amorphous or crystalline. • Non volatile. • Usually bitter in taste. • Soluble in water and polar organic solvents. • . Reduce Fehling’s solutions only after hydrolysis

11. Extraction and Isolation • Because of the wide range of physical and chemical properties of glycosides and other constituents associated with them, no common general method for their isolation is recommended. • Water, methanol, water-ethanol and ethanol are the most common solvents for extraction of glycosides. • Precautions before extraction • Deactivation of enzymes: • Drying for 15-30 min at 100 degree C followed by slow drying at a low temperature. • Dipping the fresh material into boiling water or boiling alcohol for 10-20 min. • Boiling the fresh plant material with acetone. • Carrying out the extraction at very low temp. • Freeze-drying of the plant material before extraction (lyophilization). • Carrying the extraction in the presence of (NH4)2SO4.

12. Maintenance of neutral conditions:Neutral pH should be assured before and during extraction because:Acidity may result in hydrolysis. This is overcome by addition of CaCO3. Mild alkalinity may sometimes produce racemization.Defatting of fat-rich organs (e.g. seeds) before extraction:High amounts of lipoids hinder glycoside extraction. Defatting is usually carried with petroleum ether

13. Classification • On the basis of aglycone structure • [1] Alcoholic and Phenolic Glycosides • [2] Aldehydic Glycosides • [3] Cardio-Active Glycosides • [4] Anthracene (Anthraquinone) Glycosides • [5] Saponins • [6] Cyanophore • [7] Isothiocyante • [8] Flavonoids • [9] Coumarin and Furanocoumarin • [10] Glycosidal bitters and Miscellaneous

14. Alcoholic and Phenolic Glycosides 1- Salicin • Source: Salix species (Willow bark). • Nature: Primary achholic and Phenolic glycoside (monoside). • Uses: Analgesic- Antipyretic- Anti-inflammatory.

16. 2- Arbutin & Methyl Arbutin Source: UvaUrsi (Bearberry leaves). Nature: Primary Phenolic glycoside (monoside). Uses: Diuretic- Bactericidal.

17. Aldehydic Glycosides 1- Glucovanillin • Source: Vanilla pods. • Uses: Flavouring agent- Spray reagent.

18. A small group of plant glycosides act directly on the heart muscle. These include (but are not limited to cardiac glycosides or cardenolides) Cardenolides are steroidal glycosides  exert a slowing and strengthening effect on the failing cardiac muscle. Cardio-Active Glycosides

19. Basic Structure of Cardiac Glycoside

20. Steroids in nature exerts powerful action on the cardiac muscle, as a glycoside the sugar moeity is attached to the #3 position of the steroid nucleus2 types of steroid aglyconeCardenolide (C23) – C17 β side chain -- ,  - unsaturated 5 – membered ringBufadienolide (C24) – homolog of cardenolide, C17 unsaturated 6 – memberedlactone ring - origin prototype – bufalin (Toad – Bufo sp)

23. To obtain optimum cardiac activity – aglycone must possess an ,  unsaturated lactone ring on C17 of the steroid nucleus and A/B and C/D on cis – configuration (same side substituent)Sugar – confers the molecule's solubility property, its absorption and distribution in the body (O- hydroxyl group – rapid onset of action)

24. Definition: Digitalis consists of the dried leaves of Digitalispurpurea. It is required to contain at least 0.3% of total cardinolides calculated as digitoxin. Digitalispurpurea – Purple Foxglove

25. Pharmacological Action of Cardiac Glycosides • Effectiveness depends on both the aglycones and the sugar attachments. • Medicinal action depends on the aglycone • But the sugars make the compound more soluble in increases the fixation of the glycoside to the heart muscle

26. Digitalispurpurea - Collection • There is a general belief that the pharmacological activity of the leaves increase during the course of the day to reach a maximum in the early afternoon (when they should be harvested). • This was supported by assays done in 1956 which showed a greater activity of the leaves at noon than 8am, irrespective of if the leaves where in the sun or shade. • However, other work indicates that there is no glycosidal content variation of the leaves when measured at 3-hour intervals.

27. Active Constituents • glycosides • purpurea glycoside A • purpurea glycoside B • (2 main actives in the fresh leaf) • Glucogitaloxin • At C-3 of the genin: a linear chain of 3 digitoxose sugar moieties terminated by glucose

29. Allied Drugs D. thaspi D. lutea D. ferruginia Adulterants Verbascumthapsus (mullein leaves) Symphytumofficinalis (Comfrey) Primulavulgaris (Primrose) Inulahelenium (elecampane) Urticadioica (stinging nettle) Digitalispurpurea

30. Part Used: Dried seeds (ripe) Habitat: East Africa. Greek strophos (a twisted cord or rope) anthos (a flower) Strophanthuskombe

31. Bufadienolides • Less commonly distributed in nature than cardenolides • Occur in some Liliaceae and Ranunculaceae Species. Also occur in toad venoms. • Therapeutically there is not much value as the therapeutic index is low and production of side effects high. • Squill, however has a time-honoured place as an expectorant • Widely used in the treatment of cough.

32. Bufadienolide

33. Definition: Consists of the dried sliced bulbs of Urgineamaritime, from which the membranous outer scales have been removed. It is commercially known as white squill. Description: A perennial herb growing up to 1.5 m from a large white (or red) bulb. It has a single flowering stem, a rosette of large basal leaves, and a dense spike of white flowers. Urginea maritima – (Liliaceae)

35. Cardiac Glycoside • General Tests -with FeCl3 -Foam Test • Specific Tests • -Keller-Killiani • -Salkowski • -Baljet • -Barfoed • -Seliwanoff

36. General Test: with FeCl3 .Positive Result A brown ring obtained at the interface indicates the presence of glycosides. . Principle The brown ring obtained is due to the presence of glycone sugar.

37. with FeCl3

38. Specific Tests

39. Test for Deoxy Sugars Deoxy sugars are sugars that have had a hydroxyl group replaced with a hydrogen. Keller's reagent describes its use to detect the principal components of digitalis: The reaction with this reagent is also known as the Keller-Kiliani reaction. Lack of the Keller-Kiliani reaction suggests the absence of 2-desoxysugar.

40. Specific Test: Keller-Killani Test A. Reagents 1.Glacial acetic acid 2.Ferric chloride solution 3.Concentrated sulphuric acid B. Materials and Apparatus 1.Dropping pipette 2.Test tubes C. Procedure 1.Treat five ml of each extracts with 2ml of glacial acetic acid containing one drop of ferric chloride solution. 2.Underlay the mixture with 1 ml of concentrated sulphuric acid. 3.A brown ring of the interface indicates a deoxysugar characteristic of cardenolides. 4.A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer.

41. Specific Test: Keller-Killani Test D. Positive Result Presence of brown ring on the interface and greenish ring gradually forms on the upper phase. E. Principle Involved The brown ring indicates the presence of 2-deoxysugar in the glyconeportion of the cardiac glycoside.

42. Keller-Killiani

43. Test for Steroidal Nucleus Steroids are known to perform a variety of functions in higher animals. Steroid nucleus is composed of three six member rings and one five member ring.

44. Specific Test: Salkowski A. Reagents 1. Chloroform 2. Sulfuric acid B. Materials and Apparatus 1. Dropper 2. Test tubes C. Procedure 1. Add 1ml of the extract to 2ml of chloroform. 2. Carefully add H2SO4. A reddish brown color at the interface indicates the presence of aglycone portion of cardiac glycoside.

45. Specific Test: Salkowski D. Positive Result Reddish-brown color at the interface E. Principle Involved In the Salkowski reaction based method, cholesterol is oxidized in the presence of an excess amount of phosphoric acid and ferric ions to give a reddish brown derivative. The presence of the aglycone portion of the cardiac glycoside structure is detected. The aglycone part is the portion of the structure where there is the absence of sugar. The change of color into a reddish brown at the interface indicates the aglycone portion of the corchorin.

46. Salkowski

47. Test for Lactone Ring Lactone is a cyclic ester that can be seen as the condensation product of an alcohol group -OH and a carboxylic acid group -COOH in the same molecule. It is identified by a closed ring consisting of two or more carbon atoms and a single oxygen atom, with a ketone group =O in one of the carbons adjacent to the latter. Two classes have been observed in nature - the cardenolides, which have an unsaturated butyrolactone ring and the bufadienolides, which have an a-pyrone ring.

48. Specific Test: Baljet Test • A. Reagents • 1. Picric Acid • 2. Ethanol • 3. Sodium Hydroxide • B. Materials and Apparatus • 1.Dropper • 2.Test tubes • C. Procedure • 1. To prepare Solution I for Baljet Test, place 1g of picric acid in 100mL of EtOH. • 2. To prepare Solution II for Baljet Test, add 10g NaOH in 100mL water. • 3. Combine the two solutions. • 4. Add 2-3 drops of the combined solution to 2-3mg of sample; a positive reaction is indicated by orange to deep red color.

49. Specific Test: Baljet Test D. Positive Result Orange to deep red coloration E. Principle Involved Reactions due to (-CH2-) group of the lactone ring. *cardenolide + Baljet's reagent (picric acid +NaOH) → orange or red.

50. Baljet