Useful for B.Pharmacy 5th sem

1. INDUSTRIAL PRODUCTION,ESTIMATION AND
UTILIZATION OF
PODOPHYLLOTOXIN
(BP504 T. PHARMACOGNOSY AND PHYTOCHEMISTRY )

2. SOURCE
• Podophyllotoxin (PPT), also known as podofilox,
• It is a non-alkaloid toxin lignan extracted from the roots
and dried rhizomes of Podophyllum hexandrum.
Family:Berberidaceae.
A less refined form known as podophyllum resin is also
available, but has greater side effects.

3. Chemical structure and Molecular composition
Formula C22H22O8
Molar mass 414.410 g·mol−1
Melting point 183.3 to 184 °C (361.9 to 363.2 °F)

4. Mechanism of action
• Podophyllotoxin (PDT) destabilizes microtubules by binding tubulin
and thus preventing cell division.
• In contrast, some of its derivatives display binding activity to the
enzyme topoisomerase II (TPI- II) during the late S and early G2
stage.
• It inhibits the activity and increases the level of TPI-II enzyme and
increases DNA Cleavage complex and there by leads to genetic
alteration and cell death

5. MECHANISM OF ACTION OF Podophyllotoxin PDT

6. Industrial production of Podophyllotoxin
• The use of biotechnological approaches for the production of
podophyllotoxin using cell cultures,
• Organ cultures, and
• Biotransformation route or
• By manipulating biosynthetic pathway proves to be an attractive
alternative for production of Podophyllotoxin

7. Industrial Production of podophyllotoxin
from Podophyllum hexandrum
METHODS INVOLVED IN THE PRODUCTION:
• Bio synthetic pathway
• Use of callus and cell suspension cultures for the production of
podophyllotoxin
• Use of transgenic hairy roots for the production of podophyllotoxin
• Podophyllotoxin production using biotransformation approach
• Metabolic engineering considerations for the production of podophyllotoxin

8. Bio synthetic Pathway
• Podophyllotoxin belongs to lignan group of compounds.
• Lignans are dimerization products of two phenylpropane units linked by carbon atoms of their side
chain.
• Most of the pathways proposed involve phenolic oxidative coupling of C6-C3 monomers via shikimic
acid pathway.
• Production of optically active lignan dimers is enzyme-controlled reaction.
• A series of compounds of considerable medicinal and commercial interest as clinically useful
anticancer drugs are formed by reductive dimerization of cinnamic alcohols or cinnamic acid

9. Shikimic acid Pathway

10. • Roots of P. hexandrum for the production of podophyllotoxin in
undifferentiated callus and cell suspension cultures has been reported.
• It has also been reported that coniferin was a substrate for
podophyllotoxin production
• Callus cultures of P. hexandrum Royle were very difficult to initiate.
CALLUS AND CELL SUSPENSION CULTURES

11. CALLUS AND CELL SUSPENSION CULTURES
• Cell suspensions were initiated by transferring the callus to same liquid medium
agitated on a shaker at 150 rpm.
• After ten passages homogeneous and undifferentiated suspensions were
obtained.
• Good podophyllotoxin producing cultures were dark brown coloured
• when they changed to yellow green complete loss of podophyllotoxin production
was observed.

12. Light grown callus cultures contained 3–4 times less podophyllotoxin in
comparison to dark grown cultures.
 The highest cellular accumulation of podophyllotoxin in suspension cultures
was measured at day 15 during stationary phase of growth cycle followed by
decrease in content.

13. CALLUS AND CELL SUSPENSION CULTURES
Podophyllotoxin production in callus cultures derived from rhizomes
of Podophyllum peltatum was stimulated upon illumination.
Cells cultured in media containing 4 mg L−1 Napthalene Acetic Acid
accumulated 0.001–0.002% podophyllotoxin on dry weight basis.
Feeding of 3mM coniferyl alcohol, dissolved in culture medium as β-
cyclodextrin complex resulted in enhanced podophyllotoxin
accumulation with a maximum of 0.012%.

14. Use of transgenic hairy roots for the production of Podophyllotoxin
• Genetically transformed hairy roots produced by infection of plants with
Agrobacterium rhizogenes, a gram-negative soil bacterium, appear to be a
promising tool for secondary metabolite production.
• These hairy roots are unique in their genetic and biosynthetic stability and
their fast growth offers an additional advantage.
• These hairy roots are best experimental system for production of secondary
metabolites
• Podophyllotoxins are also produced from root cultures of Linum flavum
,cultures of Linum flavum produce 5 methoxy derivatives of podophyllotoxin (5
MPT).
• Higher production (5–10 fold) of 5 MPT is reported from hairy roots than
untransformed cell suspension cultures

15. Bio-Transformation Approach
• Recently the potential of biotransformation using cell cultures for the production of etoposide has
been emphasized.
• Etoposide is derived chemically from podophyllotoxin by two step conversion.
• It has been demonstrated that a cell line of Podophyllum peltatum active in the biosynthesis of
podophyllotoxin was able to maintain repeated biotransformation by oxidative coupling of the
butanolide to the podophyllotoxin analouge.
• It has been carried out in a bioreactor for a total of more than 15 cycles each of 24 h duration
giving a yield of around 50%.
• This result indicates the feasibility of a biotransformation route to podophyllotoxin using a
chemically synthesized precursor

16. Metabolic engineering considerations for the production of podophyllotoxin
• Genetic engineering of metabolic pathways has recently been proved
to be highly beneficial for the manipulation of secondary metabolite
production.
• Podophyllotoxin, which belongs to lignan group of compounds, is the
product of Phenyl Propanoid Pathway (PPP).

17. Phenyl Propanoid Pathway (PPP)

18. Metabolic Engineering
• Recently in addition to CAD enzymes like Cinnamyl Co-enzyme
Reductase(CCR) and Laccase have been isolated which has helped in
the directed negative regulation of lignin biosynthesis.
• These developments for the reduction of lignin synthesis may offer
possibility for channeling precursor for the increased synthesis of
lignans in the branched chain in the PPP biosynthetic pathway.
• This diversion of biosynthetic pathway may offer the possibility to
increase podophyllotoxin production.

19. ESTIMATION OF PODOPHYLLOTOXIN
• A simple, sensitive, specific, rapid and accurate high performance thin layer
chromatographic (HPTLC) method has been developed for the quantification of
podophyllotoxin
• The method involved densitometric evaluation of podophyllotoxin after resolving it
on silica gel plate using dichloromethane–methanol–formic acid (9.5:0.5:0.5 v/v/v)
as the mobile phase.
• The relationship between the concentration of standard solutions and the peak
response (area) was linear within the concentration range of 150–2400 ng spot−1 for
podophyllotoxin
• The HPTLC method for the quantification of podophyllotoxin was found to be
simple, precise, specific, sensitive and accurate which can be used for routine
analysis and quality control of P. hexandrum and several formulations Instrumental
precision was found to be 1.03–1.80 (% RSD)

20. HPTLC INSTRUMENTATION AND EXPERIMENTAL CONDITIONS
• The plates were developed in a 20 × 10 cm twin trough glass chamber (Camag,
Switzerland).
• A TLC scanner III was used for scanning the TLC plates.
• Pre-coated silica gel aluminum plates 60F254 (E. Merck, Darmstadt, Germany)
with a thickness of 0.2 mm were used for all determinations.
• The plates were pre-washed with methanol and activated at 60 °C for 5 min
prior to chromatography.
• Five different volumes (0.3, 0.6, 1.2, 2.4, 4.8 μL) of standard solution of
podophyllotoxin were applied on a 20 × 10 cm TLC plate for the preparation of
the calibration curve of podophyllotoxin.
• A constant application rate of 150 nL s−1 was employed for podophyllotoxin
with a band width of 6.0mm for podophyllotoxin.
• .

21. HPTLC Instrumentation and Experimental Conditions
• The scanning speed was employed at 20 mm s−1 for podophyllotoxin and the slit
dimension was kept at 5.0 × 0.45 mm for podophyllotoxin.
• Twenty mL of mobile phase consisting of dichloromethane-methanol-formic acid
(9.5:0.5:0.5, v/v/v) was used per plate.
• The optimized chamber saturation time for mobile phase was 15 min at room
temperature (25 ± 2 °C) at relative humidity of 60 ± 5% RH.
• The plates were developed and scanned within 10 min using densitometric
scanner III in the absorbance mode at 292 nm for podophyllotoxin.
• The source of radiation was deuterium lamp emitting a continuous radiation
between 200 and 400 nm. The data obtained were analyzed by winCats software
to get linear regression equation

22. Rf value 0.66
HPTLC chromatogram of Podophyllotoxin
(a) Standard and
(b) Sample at 292 nm.

23. UTILISATION OF PODOPHYLLOTOXIN(PPT)
• Podophyllotoxin possesses a large number of medical applications, as it is able to
stop replication of both cellular and viral DNA by binding necessary enzymes.
• It can additionally destabilize microtubules and prevent cell division.
• Because of these interactions it is considered an antimitotic drug.
• Podophyllotoxin and its derivatives are used
cathartic, purgative, antiviral agent, vesicant, antihelminthic,
and antitumor agents.
• Podophyllotoxin derived antitumor agents include etoposide and teniposide.
• These drugs have been successfully used in therapy against numerous cancers
including testicular, breast, pancreatic, lung, stomach, and ovarian cancers

24. UTILISATION OF PODOPHYLLOTOXIN (PPT)
• As a potent topical antiviral.
• It is used for the treatment of HPV infections with external warts as well as
molluscum contagisum infections.
• PPT cream is highly effective with minimal side effects, which are typically
limited to itching, irritation, and redness.
• 0.5% PPT cream is prescribed for twice daily applications for 3 days followed
by 4 days with no application, this weekly cycle is repeated for 4 weeks.
• It can also be prescribed as a gel, as opposed to cream.
• PPT is also sold under the names condyline and warticon.

25. MARKETED PRODUCTS OF PODOPHYLLOTOXIN