1. Cardioactive glycosides

2. Introduction Also called cardiac glycosides.
Have specific & potent activity on cardiac muscles.
Small doses  therapeutic action.
High doses  toxic & sometimes lethal.
Plants & preparations containing cardiotonic glycosides have been used as poisons & drugs in herbal medicine since ancient times.

3. Distribution
Mainly present in : Scrophulariaceae (Digitalis), Apocynaceae (Strophanthus) Liliaceae (Urginea), & Ranunculaceae (Adonis).
Localized in different plant organs.:
seeds (Strophanthus)
leaves (Digitalis)
fruits (Acokanthera)
bulbs (Urginea= Squill)
roots (Apocynum)
herbs (Adonis)
Best source is Digitalis purpurea (purple foxglove).

4. Chemical Structure Steroidal nucleus with:
An unsaturated lactone ring attached to C-17 with -configuration
A tertiary -hydroxyl group at C-14.

5. Chemical Structure In addition to:
An axially oriented -OH at C-3 to which is attached the sugar moiety.
Methyl groups at C-10 & C-13.
Cis-fusion of rings C / D & in most cases that of rings A / B.
Other substituents on steroidal nucleus e.g.:
Replacement of -CH3 at C-10 by -CHO or -CH2OH (e.g. Strophanthus glycosides).
Additional-OHs present at C-1, C-2, C-5, C-11, C-12 & C-16.

6. Chemical Structure-Sugar moiety
Characterized by being:
Attached to C-3 of the steroidal nucleus.
Variable number of sugar units (1 – 4).
Sugars are of different types: hexoses, methyl pentoses & 2,6 deoxy hexoses etc……
Examples
Type of sugar
Glucose
Rhamnose, Fucose & Allomethylose
Digitoxose & Boivinose
Digitalose
Cymarose, Sarmentose & Oleandrose
Hexose
Methyl pentose (6-deoxy hexose)
2,6 deoxy hexose
Methyl pentose-3-methyl ether
2,6 deoxy hexose-3-methyl ether

7. Chemical Structure-Sugar moiety
Links between sugars are either 14 or 16
Generally there is no branching in the sugar chain
“Primary glycosides” have 1 or 2 molecules of glucose attached to the end of the sugar chain. Removal of these glucose units (by prolonged storage or enzymes)  “secondary glycosides”.
Sugars can modify activity (potency, toxicity), solubility, distribution & absorption of glycosides.

8. Points of differentiation
Classification
According to the type of lactone ring present in the aglycones as:
Bufadienolides
(Pentadienolide or
Scilladienolides
Cardenolides
(Butenolides)
Points of differentiation
6-membered (5 C + 1 O)
300 nm
– ve (Kedde's, Legal's &
Raymond's tests)
Squill (Urginea)
5-membered (4 C + 1 O)
220 nm
+ ve (Kedde's, Legal's &
Digitalis & Strophanthus
Structure of lactone ring
UV absorbance
Tests for identification
Examples : Glycosides of

9. Extraction Plant material + light petroleum (removal of fat)
Mark + cold water (remove soluble polysaccharide)
+ alcohol or alcohol-water mixture (extract glycosides) filter & concentrate
+ lead acetate (precipitate tannins), filter.
+ ether or chloroform + shake (extract the crude glycosides)
Chromatography (separate individual glycosides).

10. Properties, Stability & Hydrolysis
Condition: crystalline, odorless & bitter taste.
Solubility: most are hydrophobic, soluble in organic solvent, slightly soluble in water & freely soluble in alcohol.
Except: ouabain highly hydroxylated, hydrophilic & water soluble.

11. Stability- Effect of acids
Mild acidic conditions complete hydrolysis (cleavage of all glycosidic linkages)  aglycone + individual sugar units.
2-deoxy sugars directly attached to the aglycones are the most easily hydrolyzed
Drastic acidic conditions  elimination of OH group at C-14  14, 15 anhydro derivatives.

12. Stability- Effect of alkalis
Mild alkaline conditions  different products according to type of alkali:
+ NaOAc  Isomerisation of lactone ring from unstable b-oriented  stable a-oriented  inactive allo-cardenolides.
+ Na2CO3  deacylation of acylated sugars (e.g. of acetyl digitoxose in Lanatosides  corresponding Purpurea glycosides).
Drastic alkaline conditions: e.g. + strong NaOH solution  cleavage of lactone ring  carboxylic acid salt  complete loss of activity.

13. Stability- Effect of hydrolyzing enzymes
Glycosides + Enzymes  Gradual hydrolysis
Example:
Primary glycosides + a-glucosidase  removal of terminal a-glucose  Secondary glycosides

14. Chemical tests- A- Color reactions due to the aglycone moiety
Reactions due to the (-CH2-) group of the lactone ring [characteristic for 5-membered lactone ring of cardenolides]:
Legal’s test: + Na nitroprusside + NaOH deep red color.
Raymond’s test: + m-dinitrobenzene + NaOH  violet color  blue color.
Kedde’s test: + Kedde's reagents A (3, 5 dinitrobenzoic acid) & B (NaOH)  violet color.
Baljet’s test: + Baljet's reagent (picric acid + NaOH)  orange or red.

15. Chemical tests- A- Color reactions due to the aglycone moiety
Tests for steroidal nucleus: +ve with any steroidal compound including cardenolides & bufadienolides:
Antimony trichloride test: + SbCl3 / CCl4  blue or violet.
Liebermann’s test: + glacial acetic acid + 1 drop conc. H2SO4  red, violet, blue to green.

16. Chemical tests- B-Color reactions due to the sugar moiety
Keller-Killiani’s test (for 2-deoxy sugar):
Glycoside containing 2-deoxysugar + glacial
acetic acid (+ traces of FeCl3) + conc. H2SO4
(carefully added on wall of test tube 
lower layer)  blue ring between the 2
layers.

17. Quantitative determination
Cardiac glycosides must be carefully determined in crude drugs & pharmaceutical formulations as:
They are highly toxic.
Their amount in plant material is liable to variation (stage of development, seasonal variations, drying & storage conditions)

18. Methods of determination
Colorimetric: based on color tests e.g. Balget's or Kedde's.
Gravimetric.
Fluorimetric (combined with chromatography)
Biological: most widely used, based on determination of the minimum lethal dose that stops the heart under specified conditions.
Immunoassay.
Chromatographic methods e.g. RP-HPLC (Reversed Phase - High Performance Liquid Chromatography).

19. Pharmacology & mode of action
Act on the failing heart by direct or indirect effects:
Increase force of contraction (positive inotropic effect), i.e. affect tone, excitability & contractility of cardiac muscle (cardiotonic) {mechanical effect}
Slow rate of atrioventricular conduction (antiarrhythmic) {electrical effect}
Effect on the kidney: Have a diuretic effect.

20. Structure-Activity Relationship (SAR)
Cardioactivity is due to aglycone moiety.
Pharmacokinetic behavior is influenced by glycone (sugar) moiety.

21. SAR-Aglycone moiety The a, b-unsaturated lactone ring:
Was believed to be responsible for activity (but, a, b-unsaturated ester derivatives were found active)
Saturation  10 folds reduction of potency.
Opening  complete loss of activity.
The cis junction of rings C / D & A / B:
cis-junction of rings C / D is necessary for activity.
A / B cis fused rings are more potent than A / B trans.

22. SAR-Aglycone moiety
Oxygen substitution on steroidal nucleus affects distribution & metabolism.
Higher number of -OH groups  more rapid onset of action & elimination from body.
Replacement of -OH groups at C-3 & / or C-14 by H atoms  slight reduction in potency.
-orientation of C-17 side chain is necessary for activity, 17 a-cardenolides (allo-cardenolides) are inactive.

23. SAR-Sugar moiety
Sugar moiety has no cardiac activity, but when attached to C-3 OH group of steroidal moiety  modify activity & influence pharmacokinetic behavior.
Free aglycones are less potent than their glycosides.
Type & number of sugar units may affect potency.
Cardiac glycosides with 6-deoxy sugars (6-CH3) are more potent than their 6-CH2OH analogs.
For oligosaccharide moieties: potency decreases with increase in number of sugar units.

24. Therapeutic uses Indicated for various cardiac conditions:
Congestive heart failure (elderly people).
Atrial tachyarrhythmia (atrial fibrillation).

25. Administration
Careful selection of drug due to difference in onset & duration of action.
Duration of action may be:
Long : (Digitoxigenin-derived glycosides)
Intermediate: (Digoxigenin-derived glycosides)
Short: (Strophanthus glycosides, used by i.v. injection only in acute congestive heart failure)
Administered in an initial loading dose (in order to bring the heart under the influence of the drug) followed by a daily maintenance dose

26. Elimination
Digitalis purpurea glycosides are more cumulative than those of D. lanata.
Strophanthus glycosides (e.g. ouabain) have a rapid onset of action & elimination.

27. Digitalis Glycosides - Source
Digitalis spp (Scrophulariaceae)
D. lanata (total glycosides, 1.5 %) with major: Lanatosides A-E (primary glycosides) & digoxin.
D. purpurea (total 0.5 %) with major : Purpurea glycosides A, B & E, digitoxin, gitoxin & gitaloxin.

28. Digitalis lanata glycosides - Structure
Primary glycosides with acetylated sugar moieties.
Major constituents:

29. Digitalis purpurea glycosides
About 30 glycosides derived from digitoxigenin, gitoxigenin & gitaloxigenin
Not acetylated at the 3-position of the terminal digitoxose unit.
Major: Purpurea glycosides A, B & E.
Gitaloxin is the most active. On storage it is converted to gitoxin

30. Digitoxin
Lanatoside A
Digitoxin

31. Digoxin (Lanoxin®)
Lanatoside C
Digoxin

32. Comparison between Digoxin & Digitoxin
Most liposoluble cardiac glycoside
Digoxin (Lanoxin®)
Name
D. purpurea, & lanata
D. lanata.
Source
Digitoxigenin+3 Digitoxose
Digoxigenin + 3 digitoxose
Hydrolysis
Oral
Usually oral
Administration
After 1-4 hours
After 30 min to 2 hours
Onset of action
At hours
At 2 to 6 hours.
Peak
168 to 192 hours
30 to 40 hours
Plasma half-life
3 to 5 weeks
6 to 8 weeks
Complete elimination
after discontinuation of
therapy
Eliminated through liver so recommended for patients with impaired renal function.
Eliminated through kidney
Elimination
14-26 ng / ml
0.5-2ng / ml
Full therapeutic effect
35 ng / ml
2.5 ng / ml
Toxicity symptoms
Recommended for
patients with impaired
renal function.
When risk of intoxication
is great, as it is relatively
short-acting&rapidly eliminated
Indication

33. Strophanthus glycosides
Dried, ripe seeds of Strophanthus kombe, S.
gratus & S. hispidus (Fam. Apocynaceae)
(used in Africa as arrow poison)
Strophanthus gratus
Strophanthus kombe

34. Strophanthus kombe glycosides

35. K-strophanthoside (Stroposide)
Source:
Principal primary glycoside in S. kombe & S. hispidus.
Hydrolysis:
Enzymatic (gradual)
K-strophanthoside+ -Glucosidase  terminal -glucose + K-strophanthin-B
K-strophanthin-B + strophanthobiase  -glucose + cymarin
Uses:
K-strophanthin-B (like ouabain) is mainly used for intravenous therapy.
Chemical tests:
+ 66% H2SO4  emerald green color.
Solution in H2O + FeCl3 + H2SO4  red color  green color.

36. Strophanthus gratus glycosides- Ouabain (G-strophanthin)
Source: Seeds of S. gratus (Apocynaceae).
Hydrolysis: ouabagenin + rhamnose.
Structure:
Most polar cardiac glycoside.
Characterized by the presence of -CH2OH group at C-10 & additional OH groups at C-1& C-11.
Chemical test
+ 66% H2SO4  pink color  green fluorescence.
+ Froehd’s reagent, evaporate + conc. H2SO4  blue color.
Uses
Used as cardiotonic & antiarrhythmic
agent.
Ouabain

37. Squill glycosides - White squill
White squill = Medicinal squill
Squill or squill bulb = cut & dried, fleshy, inner scales or bulb of white variety of Urginea maritima (Mediterranean squill) or of U. indica (Indian squill, Liliaceae).
Contains 0.3 % of mixture of glycosides: scillaren A (major), scillaren B, urginin A & B, & scilliphaeoside
Proscillaridin A is the therapeutically used bufadienolide glycoside obtained by enzymatic hydrolysis with the endogenous enzyme scillarenase.

38. Squill glycosides- White squill
Chemical test
Squill glycosides give positive tests for the steroidal moiety only, as they neither contain a pentacyclic lactone ring with a -CH2 group (c.f. cardenolides) nor a 2- deoxy sugar in their sugar moiety.
Squill glycoside or aglycone + acetic anhydride + H2SO4 blood red  blue  bluish green color.
Uses
Expectorant & emetic (mainly due to saponin), cardiotonic & diuretic.
Cardiac glycosides of squill exert a rapid cardiotonic action & are rapidly eliminated & less potent than other cardiac glycosides.

39. Red squill
Bulb & bulb scales of red variety of U. maritima, used as rat poison (rodenticide)
Should not be present in medicinal squill
Detected by presence of red, pink, or purple epidermal or parenchymatous tissues.