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

• 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 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

tautomers11 keto diosgenin

Marker

11 keto progesterone

hydrocortisone