Plant steroids Anna Drew with grateful acknowledgement for

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

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)

Structure • Hydrocarbons – (3 x 6 C) + (1 x 5 C) = tetracyclic triterpenoid type – ring junctions sometimes contain 3 y methyl groups – normally side chains are at C 17 (classified by this) – and functional groups at C 3 (-O or -OH groups) – also at C 11 (-O, –OH gives oxygen function)

– shape very important for biological activity – 3 D determined by ring junction AB – CD Trans AB isomer Cis AB isomer • 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

Hormones – plant sources • GROUP 1 sapogenins • GROUP 2 phytosterols • occur as glycosides linked • occur as ester linked to to a sugar fatty acids • polar, soluble in alcohol • non-polar, soluble in and alcohol/water hexane and petroleum mixtures spirits • occur in leaves -> roots, • occur in fruits and seeds rhizomes Occur in very large amounts in plants – 10 -25% by weight of plant material

Sapogenins • Sapogenin = steroid nucleus • Saponin = glycosides + sugars – ‘soap-like’ in nature – have been used to poison fish • accumulates in gills preventing O 2 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

• 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

[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 • Digitalis seeds full of steroids, rich source [6] Leguminosae • Trigonella-foeum-graecum fengreek seed

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 C 17

• 2 isomers at C 25 due to free rotation around it – no other isomers occur naturally 25 α iso-series 25 β neo-series • sugars attach at C 3 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 water|oil or air|water interface

Tigogenin Structure courtesy of www. chemblink. com – – simplest sapogenin has correct configuration from which to make steroids occurs with the isomer neotigogenin widely distributed in plants: • yam, digitalis seeds, fenugreek seeds

Diosgenin Structure courtesy of www. chemblink. com – 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

Hecogenin Structure courtesy of www. chemblink. com – from the sisal plant, various species of yucca • Philipines and Far East – – isomer is sisalgenin keto function at C 12 important corticosteroids have C 11 =O group giving activity here C 11 cannot be substituted; C 12 =O enables halogenation at C 11, then =O removed at C 12

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, Me. OH, H 2 O

• plant material dried in an oven • Soxhlet extraction with petroleum spirit – to distill over saponins – crystallise out in receiver – 10 g/100 g 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 (H 2 O, HCl) – petroleum spirit can be recovered • recrystallisation expensive but gives a high yield • p’ceutical companies will buy compounds in pure

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 – 960 cm-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

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. .

• 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) couldn’t 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

[2] Hecogenins – more economical 11 keto progesterone hydrocortisone Marker 11 keto diosgenin tautomers – NB Diosgenin can be used to produce hydrocortisone but a fermentation stage is needed to introduce O- at C 11 keto α position of pregnane nucleus