Plant steroids

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

2. 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)

3. 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)

4. 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

5. 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 Hormones – plant sources Occur in very large amounts in plants – 10-25% by weight of plant material

6. 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

7. 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

8. [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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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....

19. 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

20. [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