Cardiac glycosides Flavonoids

1. Cardiac glycosidesFlavonoids Anna Drew with grateful acknowledgement for inspirational teaching received at The School of Pharmacy, University of London

2. Cardiac glycosides • Plant glycosides with specific action on heart • Historical use: • to assasinate people, arrow poisons • Historical sources: • South American toad skins, African plant extracts • Modern use: • to treat congestive heart failure (dropsy) • aglycone structure important for activity

3. Sources • Scrophulariaceae • Digitalis purpurea leaves (foxglove) • Digitalis lanata leaves – white flowers • Apocyanaceae • Strophanthus vine seeds – Africa • Liliceae • Urginea bulbs (squill) – Europe, India • Convallaria leaves (lily of the valley) – also produces a volatile oil perfume

4. Active compounds • steroid nucleus • AB cis-junction • CD cis-junction • not planar • C14 = 3y -OH • C3 = 2y –OH • sugars attached • C17 = lactone ring • classified into 2 groups Cardenolides more common opens in alkali Bufanolides

5. sugars: • 1-4 β-linked at C3 in various combinations • glucose, rhamnose, deoxy-sugars • eg digitoxose, digitalose StrophanidiolStrophanthus Scillarenin (squill)

6. Extraction • large molecular weight molecules with sugars -> polar • soluble in water and alcohol • expensive – long process, solvents

7. Digitalis • Scrophulariaceae family • foxglove - biennial flowering plants • cases of poisoning rare • natural emetic if eaten in excess • Digitalis purpurea leaf – purple, British • -> Digitalis Tablets B.P. • -> Tincture of Digitalis B.P. • commercially grown Holland, E. Europe • NB no extraction for these products • Digitalis lanata leaf – white, Mediterranean • used for manufacture of pure glycosides • ie digoxin, lanatoside C • commericially produced Holland, Equador, USA

8. Chemistry of D.lanata • compounds belong to cardenolide series • 5 membered lactone ring • approx 96 compounds • [1930-1950 Stroll worked on structures] * * Acetyl group confers crystalline properties - makes compounds more easily isolated

9. [i] Digitoxose • sugar found on primary glycosides of D.lanata • glucose on the end of a chain of O-linked digitoxose sugars at C3 • during harvesting and drying enzymes can remove acetyl groups and the end glucose • hence drying method needs to be followed or glycosides degrade further • after collection dried as rapidly as possible at 60oC, stored in airtight containers protected from light (contain no more than 6% moisture) • expect about 10 compounds from D.lanata • important ones: • Digoxin “Lanoxin” – Wellcome – 0.25 μg white tablet • Digitoxin “Digitalin” 0.25 μg small pink tablet • Lanatoside “Cedilanid” 0.10 μg – less well absorbed but used for rapid digitalisation • Others not marketed, used experimentally

10. Some cardioactive glycosides from D.lanata: Ref: Trease & Evans

11. Chemistry of D.purpurea • Steroid cardenolides • contains 30 glycosides, 6 main ones • only has 3 aglycones • Purpurea 1y glycosides • do NOT have acetylated digitoxose third sugar • but these are found in smaller quantities • called ABE series

12. [ii] Digitalose • found in both species • only strospeside important as emergency injection for heart attacks – quickest acting cardiac glycoside

13. Assay of Digitalis B.P. • required to contain not less than 0.3% total cardenolides calculated as digitoxin • important to guarantee reproducibility of products (drug dosage) • narrow therapeutic index • can cause cardiac arrest • slowly excreted, bound to serum proteins • long term therapy for patients • patients tend to be older, weak

14. Digitalis B.P. tablets • crushed dried leaves -> green tablet • contain 30 glycosides each with different onset, action and excretion profiles • in different amounts • influenced by growing conditions • (temp, water, sun, drying process) • assay for each glycoside as accurately as possible – dilute effects by adding grass • Two ways:

15. [1] Biological assay • British method - inaccurate but safer • Tincture of extract of leaves or tablets • diluted with saline so alcohol <6%v/v • guinea pigs (6 test, 6 control) x 3 =36 • expensive but can average results • measure volume injected into vein of leg/foot before heart stops beating • monitor heart rate via ECG • or open chest wall and watch inserted needle with flag on move • Better to watch ECG – have to differentiate from death from too large an injected volume • trained staff required, can calculate potency • assay acceptable within 80-120% error margin (not that accurate)

16. Disadvantages • inaccurate, expensive • injecting material IV (avoiding absorption, excretion) • end point is death • toxicity test not therapeutic assessment • Advantages • assessing some biological activity • safety mechanism

17. [2] Chemical assay • Problem: 30 different glycosides – can measure them accurately but may not correlate with therapeutic activity of drug • Make a tincture (with alcohol) • decolourise with lead subacetate • extract glycosides by partition with CHCl3 • evaporate to give residue (containing cardiac glycosides) • hydrolyse with HCl to remove sugars leaving aglycone • residue contains gitoxigenin and digitoxigenin (AB series) • gitaloxigenin -> gitoxigenin when acid hydrolysed

18. Colourimetric assay to separately determine material amounts (i) total aglycone • purple colour with dinitrobenzoic acid and alkali (ii) digitoxigenin only • green colour with FeCl3 + acetic acid • can substract answers to work out • Digitoxigenin (A series) content • Gitoxigenin (B series) content • Advantages: • precise method (reproducible 2%, standard error 5%) • unqualified staff, quicker • Disadvantages: • doesn’t correlate with biological activity • only estimating approx 60% therapeutic material • BOTH methods used in industry

19. Flavonoids • mainly O-linked glycosides • occur in plants, lichens, moss • those in free state and glycosides largest naturally occurring group of phenols • aromatic, based on γ-pyrone moiety • can get several forms of flavonoids depending on nuclei flavonol isoflavone flavone

20. often yellow (flavus Latin – yellow) • known for a long time • interest in them for • anti-inflammatory (and analgesic) properties • anti-allergic effects • antithrombotic, vasoprotective properties • decrease capillary fragility • phlebitis – changes in vessel walls in extremities -> plasma leakage -> oedema • mainly due to high oestrogen, sometimes in males • tumour inhibition promotion • protective for gastric mucosa

21. sugars: • glucose, rhamnose, arabinose, xylose • 2-3 attached to phenolic groups in middle of structures

22. Examples (a) Rutin (Vitamin P) • from Fagopyrum esculentum (buckwheat) = rhamnoglucoside of quercetin (b) Hesperidin (‘citrin’) • from citrus industry = hesperetin (methyl eriodictyol), rhamnose, glucose

23. Isolation • easy • water and alcohol soluble • give brightly coloured solutions • crystallise easily • may give colour reactions eg • MgCl2 -> violet -> orange • alkali KOH -> orange • easy to detect

24. Coumarins • aromatics based on α-pyrone • widely distributed in plants • Leguminosae • Rubiaceae • Umbelliferae • Solanaceae • first medicinal compounds from clover • certain types toxic to animals in summer • anticoagulant activity found • dicoumarols produced clinically