Plant steroids

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Plant steroids

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

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PLANT STEROIDS

• Used for
• replacement therapy (male female)
• athletes (glucocorticoids)
• skin conditions (hydrocortisone)
• antifertility pill (oestrogens progesterones)
• cancer (breast, testes, prostrate)
• rheumatoid arthritis
• Industrial demand may be met by plant sources or
  replaced by synthetic sources (expensive)

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Structure

• Hydrocarbons
• (3 x 6C) (1 x 5C) tetracyclic triterpenoid
  type
• ring junctions sometimes contain 3y methyl groups
• normally side chains are at C17 (classified by
  this)
• and functional groups at C3 (-O or -OH groups)
• also at C11 (-O, OH gives oxygen function)

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• shape very important for biological activity
• 3D determined by ring junction AB CD
• AB-CD trans junction tend to have a flat, planar
  structure
• important for hormonal activity
• AB-CD cis junction are bent or buckled
• allows them to fit on heart / smooth muscle and
  blood protein receptor sites
• poisonous steroids some used in heart disease

Trans AB isomer
Cis AB isomer
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Hormones plant sources

• GROUP 1 sapogenins
• occur as glycosides linked to a sugar
• polar, soluble in alcohol and alcohol/water
  mixtures
• occur in leaves -gt roots, rhizomes

• GROUP 2 phytosterols
• occur as ester linked to fatty acids
• non-polar, soluble in hexane and petroleum
  spirits
• occur in fruits and seeds

Occur in very large amounts in plants 10-25
by weight of plant material
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Sapogenins

• Sapogenin steroid nucleus
• Saponin glycosides sugars
• soap-like in nature
• have been used to poison fish
• accumulates in gills preventing O2 transfer
• also frogs and toads
• breathe through skin and hence are killed
• not poisonous to mammals when eaten
• not absorbed in intestine or stomach
• may irritate bowel causing diarrhoea
• few effects

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• if injected different
• used in arrow poisons
• cause haemolysis of red blood cells
• breaks down red blood cell membrane
• haemoglobinuria
• some used as emulsifying agents
• interested in the aglycone from a saponin
• Saponins occur widely in plants
• some economically important ones
• Sources
• 1 Dioscoreaceae (yam family)
• Dioscorea genus dicots vines
• sweet yam food source, very low steroid content
• bitter yam Mexico, South America high content

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• 2 Liliaceae family
• monocots Far East, Phillipines
• Smilax or Yucca
• very important since these provide sapogenins for
  manufacture of corticosteroids
• 3 Amaryllidaceae
• Agave sisalana sisal leaf, East Africa
• 4 Solanaceae
• can be used when supply of 1 and 2 short or
  too expensive
• Solanum sp. contain steroidal saponins
• as well as tropane alkaloids, atropine, etc
• eg tomato, potato, woody nightshade
• 5 Scrophulariaceae

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• Structure
• based on steroid nucleus
• flat trans- shape
• right shape steroid to make hormones
• occurs in a high concentration in plants
• spiroketal side chain easily oxidised off (leaves
  unstable progesterone)
• spiroketal side chain at C17

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• 2 isomers at C25 due to free rotation around it
• no other isomers occur naturally
• sugars attach at C3 to make sapogenins saponins
• tend to have quite large molecular weight
• eg 3-12 sugars polysaccharide side chain
• common sugars xylose, galactose, rhamnose,
  glucose
• combination of these sugars is usually a branched
  complex structure with high mol wt (ie not linear
  sugar chains)
• lipid soluble steroid part water soluble sugar
  part
• can orientate at wateroil or airwater interface

25 a iso-series
25 ß neo-series
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Tigogenin

• simplest sapogenin
• has correct configuration from which to make
  steroids
• occurs with the isomer neotigogenin
• widely distributed in plants
• yam, digitalis seeds, fenugreek seeds

Structure courtesy of www.chemblink.com
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Diosgenin

• can obtain prednenolone and progesterone from it
• occurs with isomer yamogenin
• occurs with some tigogenin in
• fenugreek seeds and Mexican wild yam Dioscorea
  mexicana (and Japanese types)
• hard to cultivate yams tubers underground may
  take years to grow large enough mostly taken
  from wild

Structure courtesy of www.chemblink.com
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Hecogenin

• from the sisal plant, various species of yucca
• Philipines and Far East
• isomer is sisalgenin
• keto function at C12 important
• corticosteroids have C11 O group giving activity
• here C11 cannot be substituted C12 O enables
  halogenation at C11, then O removed at C12

Structure courtesy of www.chemblink.com
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Commercial extraction

• sources crushed
• tubers yams seeds fenugreek, digitalis
  leaves sisal
• fermentation
• add excess water in fermenting vat and leave
  24-48 hour
• saponins are covalently bonded into cellulose
  wall
• own enzymes act on the polysaccharides in the
  cell wall to liberate them
• filtration to collect plant powder
• acid hydrolysis to split off saponins from
  sapogenins
• equal HCL, MeOH, H2O

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• plant material dried in an oven
• Soxhlet extraction with petroleum spirit
• to distill over saponins
• crystallise out in receiver
• 10g/100g yam tuber high yield economic
• recrystallise
• using various solvents depending on desired
  compound
• can be carried out on a large scale
• cheap
• no chromatographic process
• materials cheap (H2O, HCl) petroleum spirit can
  be recovered
• recrystallisation expensive but gives a high
  yield
• pceutical companies will buy compounds in pure

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Analysis of plant material

• Qualitative
• TLC on sulphuric acid to indicate spot position
  (chloroform solvent)
• Quantitative
• i) colorimetric assay sulphuric acid produces
  orange colour with steroids
• ii) IR spectrometry 960cm-1
• need a lot of plant material
• iii) GLC micromethod draw up assay with
  suitable standard and do many samples in one day
• quickest
• qualitative and quantitative

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Commerical use

• production of steroids from diosgenin
• before 1940 isolate from animal glands or urine
  expensive
• 1940 Marker (USA) discovered a process
  essentially same process is still used
• diosgenin extracted from Mexican yam
• then spiroketone chain is opened up....

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• in theory process gave 100 yield
• progesterone known to prevent ovulation tried
  to produce the pill (1950)
• now have combination pills
• 1950-1960 corticosteroids needed
• antiinflammatory, anticancer, antirheumatoid
• hydrocortisone and cortisone (which can be
  fluorinated) couldnt be produced from
  progesterone
• 1 Fermentation
• arose by accident when making antibiotics from
  Rhizopus
• needed steroids in medium to grow
• produced 11 keto progesterone
• analysed fermentation to confirm pregnenolone /
  progesterone were producing 11 keto progesterone
  (from which hydrocortisone can be made)
• biotechnology expensive

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• 2 Hecogenins
• more economical
• NB Diosgenin can be used to produce
  hydrocortisone but a fermentation stage is needed
  to introduce O- at C11 keto a position of
  pregnane nucleus

11 keto progesterone
Marker
tautomers
11 keto diosgenin
hydrocortisone