1. Cardiac glycosides Flavonoids
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 Apocyanaceae Liliceae
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
StrophanidiolStrophanthus
Scillarenin (squill)
sugars:
1-4 β-linked at C3 in various combinations
glucose, rhamnose, deoxy-sugars
eg digitoxose, digitalose

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
[ Stroll worked on structures] * R1 R2
Names 1y 2y H
digitoxigenin A
digitoxin OH
gitoxigenin B
gitoxin
digoxigenin C
digoxin
diginatigenin D
diginatin
formylester
gitaloxigenin E
gitaloxin
* 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
Aglycones 1y 2y
digitoxigenin A
digitoxin
gitoxigenin B
gitoxin
gitaloxigenin E
gitaloxin

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 Two ways:
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 % error margin (not that accurate)

16. Disadvantages Advantages 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) (b) Hesperidin (‘citrin’)
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