Cardiac glycosides Flavonoids

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Cardiac glycosidesFlavonoids

• Anna Drew
• with grateful acknowledgement for inspirational
  teaching received at
• The School of Pharmacy, University of London

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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

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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

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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
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StrophanidiolStrophanthus
Scillarenin (squill)

• sugars
• 1-4 ß-linked at C3 in various combinations
• glucose, rhamnose, deoxy-sugars
• eg digitoxose, digitalose

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Extraction

• large molecular weight molecules with sugars -gt
  polar
• soluble in water and alcohol
• expensive long process, solvents

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Digitalis

• Scrophulariaceae family
• foxglove - biennial flowering plants
• cases of poisoning rare
• natural emetic if eaten in excess
• Digitalis purpurea leaf purple, British
• -gt Digitalis Tablets B.P.
• -gt 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

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Chemistry of D.lanata

• compounds belong to cardenolide series
• 5 membered lactone ring
• approx 96 compounds
• 1930-1950 Stroll worked on structures

R1 R2 Names 1y 1y 2y
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
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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

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Some cardioactive glycosides from D.lanata
Ref Trease Evans
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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
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ii Digitalose

• found in both species
• only strospeside important as emergency injection
  for heart attacks quickest acting cardiac
  glycoside

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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

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• Digitalis B.P. tablets
• crushed dried leaves -gt 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

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1 Biological assay

• British method - inaccurate but safer
• Tincture of extract of leaves or tablets
• diluted with saline so alcohol lt6v/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)

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• 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

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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 -gt gitoxigenin when acid hydrolysed

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• 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
• doesnt correlate with biological activity
• only estimating approx 60 therapeutic material
• BOTH methods used in industry

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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 ?-pyrone moiety
• can get several forms of flavonoids depending on
  nuclei

flavonol
isoflavone
flavone
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• 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
• -gt plasma leakage -gt oedema
• mainly due to high oestrogen, sometimes in males
• tumour inhibition promotion
• protective for gastric mucosa

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

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Examples

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

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Isolation

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

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• Coumarins
• aromatics based on a-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