Transcript Cardiac glycosides

Cardiac glycosides
Flavonoids
Anna Drew
with grateful acknowledgement for inspirational teaching received at
The School of Pharmacy, University of London
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
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
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
Strophanidiol
Scillarenin
Strophanthus
(squill)
• sugars:
– 1-4 β-linked at C3 in various combinations
– glucose, rhamnose, deoxy-sugars
• eg digitoxose, digitalose
Extraction
• large molecular weight molecules with sugars -> polar
– soluble in water and alcohol
• expensive – long process, solvents
Digitalis
• Scrophulariaceae family
– foxglove - biennial flowering plants
• cases of poisoning rare
• natural emetic if eaten in excess
– Digitalis purpurea leaf – purple, British
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-> 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
Chemistry of D.lanata
• compounds belong to
cardenolide series
– 5 membered lactone ring
– approx 96 compounds
• [1930-1950 Stroll
worked on structures]
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1y
2y
R1
R2
Names
H
H
digitoxigenin
A
A
digitoxin
H
OH
gitoxigenin
B
B
gitoxin
OH
H
digoxigenin
C
C
digoxin
OH
OH
diginatigenin
D
D
diginatin
H
formylester
gitaloxigenin
E
E
gitaloxin
* Acetyl group
confers crystalline
properties - makes
compounds more
easily isolated
[i] Digitoxose
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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)
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expect about 10 compounds from D.lanata
– important ones:
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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
Some cardioactive glycosides from D.lanata:
Ref: Trease & Evans
Chemistry of D.purpurea
• Steroid cardenolides
– contains 30 glycosides, 6 main ones
– only has 3 aglycones
Aglycones
1y
2y
digitoxigenin
A
digitoxin
gitoxigenin
B
gitoxin
gitaloxigenin
E
gitaloxin
• Purpurea 1y glycosides
– do NOT have acetylated digitoxose third sugar
• but these are found in smaller quantities
– called ABE series
[ii] Digitalose
• found in both species
• only strospeside important as emergency injection for heart attacks –
quickest acting cardiac glycoside
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)
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narrow therapeutic index
can cause cardiac arrest
slowly excreted, bound to serum proteins
long term therapy for patients
patients tend to be older, weak
• 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:
[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)
• 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
[2] Chemical assay
• Problem: 30 different glycosides – can measure them
accurately but may not correlate with therapeutic activity
of drug
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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
• 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
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
flavone
isoflavone
flavonol
• 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
• sugars:
– glucose, rhamnose, arabinose, xylose
– 2-3 attached to phenolic groups in middle of
structures
Examples
(a) Rutin (Vitamin P)
– from Fagopyrum esculentum
(buckwheat)
= rhamnoglucoside of quercetin
(b) Hesperidin (‘citrin’)
– from citrus industry
= hesperetin (methyl eriodictyol),
rhamnose, glucose
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
Coumarins
– aromatics based on α-pyrone
– widely distributed in plants
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Leguminosae
Rubiaceae
Umbelliferae
Solanaceae
– first medicinal compounds from
clover
• certain types toxic to animals in
summer
• anticoagulant activity found
• dicoumarols produced clinically