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 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)

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

Hormones – plant sources GROUP 1 sapogenins occur as glycosides linked to a sugar polar, soluble in alcohol and alcohol/water mixtures occur in leaves -> 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

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

some used as emulsifying agents 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 [3] Amaryllidaceae [4] Solanaceae 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 C17

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 water|oil or air|water interface 25 α iso-series 25 β neo-series

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

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

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

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

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 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 – 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

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 NB Diosgenin can be used to produce hydrocortisone but a fermentation stage is needed to introduce O- at C11 keto α position of pregnane nucleus 11 keto progesterone Marker tautomers 11 keto diosgenin hydrocortisone