Cardioactive glycosides

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.

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).

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

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.

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

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.

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

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).

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.

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.

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.

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

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.

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.

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.

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)

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).

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.

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

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.

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.

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.

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

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

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

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.

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

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

Digitoxin Lanatoside A Digitoxin

Digoxin (Lanoxin®) Lanatoside C Digoxin

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 8 - 14 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

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

Strophanthus kombe glycosides

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.

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

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.

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.

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.