Industrial production of Diosgenin, digoxin and podophyllotoxin by plant tissue culture and total synthesis

1. Pharmacognosy
Rajasekhar Reddy Alavala
M. Pharm., (PhD)
Assistant Professor,
K L COLLEGE OF PHARMACY, Vaddeswaram, Guntur, A.P-
522502

2. • Diosgenin, a precursor for partial synthesis of oral
contraceptives, sex hormones, and other steroids, is
widely used in pharmaceutical industry.
• Nowadays, many researchers found that diosgenin has
antiproliferative and proapoptotic effects on cancer cells
or on rheumatoid arthritis synoviocytes.
• It also shows pharmacological activities such as
antilipoperoxidative and antiskin aging effects.
• The main raw material used in industry is Dioscorea
zingiberensis C. H. Wright (DZW) because of the high
content of diosgenin in its tubers.

3. Plant material:
• Seeds were surface sterilized with a sodium
hypochlorite solution (1.5% w/v available
chlorine) supplemented with two drops Triton X-
100 for 6 min and rinsed 3 times with sterile
distilled water.
• They are left for germination on agar plates
containing 0.5% sucrose at 25°C in the light with a
daily 16-h photoperiod.
• Plantlets are transferred onto solid half-strength
McCown’s woody plant (WP) medium (Lloyd and
McCown 1980) +3% sucrose (1/2 WP 3) and
maintained at 25°C in the light.

4. Establishment of hairy root cultures:
• Hairy roots are induced by direct infection of the stems of 2-
weekold sterile plantlets with A. rhizogenes A4. Approximately 4
weeks after infection, hairy roots were transferred onto solid 1/2
WP 3 medium containing 0.25 g/l cefotaxime and 1 g/l
ampicillin.
• After elimination of the bacteria, the hairy roots are inoculated
into 1/2 WP 3 liquid medium (50 ml in 250-ml conical flasks),
cultured in darkness at 25°C on a gyratory shaker (80 rpm) and
subcultured at 4-week intervals.
• To prove transformation, the opines are extracted and identified
by paper electrophoresis.

5. Extraction of diosgenin:
• The contents of flasks are harvested, and fresh and dry
weight, after lyophilization, will be determined
individually.
• The hairy roots are powdered, extracted and hydrolyzed by
refluxing for 5 h with 1 M sulfuric acid in 70%
isopropanol.
• The extract is adsorbed on an Extrelut column and
diosgenin was eluted with n-hexane.
• The organic solvent is evaporated to dryness. Root tissues
of normal 8-month-old plants grown in the field are
extracted.
• After filtering off cell debris, the liquid media are
lyophilized and extracted using the same procedure.

6. • Cellulase
• Pectinase and
• Glucoamylase

7. • Diosgenin was identified by high-performance liquid chromatography coupled with mass
spectrometry and comparison with reference material.
• 2 ml of dried pyridine and 0.1 ml of benzoyl chloride were added to the dried extract.
• The sample was heated at 80°C for 30 min then, after addition of 2 ml of methanol, heated again
at 100°C for 30 min.
• After cooling to room temperature, 10 ml of dichloromethane was added, followed by 20 ml of
water and 2 ml of concentrated hydrochloric acid.
• The organic phase was washed successively with water, with a saturated sodium carbonate
solution, and finally twice with water.

8. • The dichloromethane layer was evaporated to
dryness and the residue was dissolved in chloroform
(HPLC grade) before injection into the HPLC
column.
• Vitamin K1 was used as internal standard. Twenty
microliters of filtered extract-internal standard
mixture was injected. Diosgenin benzoate
concentrations were determined by comparison with
an external standard curve over the range 25–250
µg/ml.
• HPLC analyses were performed using a Waters 600E
apparatus on a Nucleosil 100-5 C-18 column fitted
with a Nucleosil 120-5 C-18 guard column.
• The isocratic solvent system was acetonitrile-water
92:8 (v/v) for 60 min with UV detection at 230 nm.
The flow rate was set at 2 ml/min and the column
temperature was maintained at room temperature.

9. • Cell culture conditions: Stock suspension cultures of the
Digitalis lanata cell line W.1.4 were grown in Erlenmeyer
flasks kept in the dark at 24°C on gyratory shakers (120 rpm).
Cells were subcultured every 10.5 d by inoculating 20 g cells
(wet weight) into 300 ml of fresh GM1
• Growth media: The maintenance medium (GM1) was based
on MS medium with twice the MS phosphate and glycine and
no caseine hydrolysate added.
• Phytohormones were omitted and the glucose concentration
was 3%. Growth medium 2 (GM2), with increased
concentrations of sulfate, phosphate, ammonium, magnesium,
potassium and glucose, was used to supply fresh medium
when the cells were grown in the semi-continuous mode.

10. • Production media: Production medium 1 (PM1), i.e., an 8% glucose solution with the pH
adjusted to 5.5, was used as the production medium for all experiments run in the batch mode. For
the production of digoxin under semi-continuous culture conditions a medium termed PM 3 (16%
glucose solution, pH 5.5) was used to replace part of the GM 2 at the beginning of the pre-
incubation phase.
• Growth of cell supension cultures in bioreactors. The contents of 24 stock culture flasks (for a
total of 7.7 l suspension) were added to 28 1 GM 2 in a 40-1 air-lift bioreactor, which was used to
produce the inoculum for a 300-1 bioreactor.

11. Production of digoxin in bioreactors:
• In preliminary runs digoxin production was achieved in a 1-1 exsiccator vessel fitted with an
aeration line ending in a ring-shaped sparger fixed to the bottom of the jar.
• The vessel was filled with 300-400 ml of cell suspensions pre-incubated for 48 h in an 8% glucose
solution. The suspension was agitated by sparged air at an aeration rate of 1 1 min-1.
• These portable glass jars were sterilized in an autoclave and then each filled with 18-19 L of cell
suspension withdrawn from the 300-L bioreactor. During incubation the glass vessels were shaded.
• The suspensions were aerated at 4.5-12.0 1 min with sterile air and the incubation temperature was
maintained at 21°C. The production cycle was started by the addition of 0.65 mmol 1-1 digitoxin.

12. Analysis:
• HPLC

13. TOTAL SYNTHESIS