Major secondary metabolites derieved for treatment of Cancer and the strategies to improve the productivity process.

1.  Organ cultures for secondary metabolite production
- In vitro organ culture of Fritillaria unibracteata rapidly propagates, directly
from small cuttings of the bulb. The growth rate of 30–50 times higher than that
under natural wild growth conditions is seen. The content of alkaloid and
beneficial microelements in the cultured bulbs were higher than found in the wild
bulb.
 Precursor addition for improvement of secondary metabolite
production
- Coniferyl alcohol in the form of complex with β-cyclodextrin was used as
precursor for podophyllotoxin accumulation in P. hexandrum cell suspension
cultures.
- The involvement of amino acids (Val, Ile) in the biosynthesis of hyperforin and
adhyperforin was reported in H. perforatum shoot cultures.
 Elicitation of in vitro products
- The addition of selenium for ginseng production.
- Chitosan, a biotic elicitor polysaccharide increases production of anthraquinone
in Rubia akane cell culture.

2.  Hairy root cultures as a source of secondary metabolites
- Inoculation of plant with Agrobacterium rhizogenes produces infection in roots,
exuding out the secondary metabolites with higher yields.
 Genetic manipulation in hairy root culture for secondary metabolite
production
- Hairy root cultures of Nicotiana rustica with ornithin decarbosylase gene from
yeast, and Peganum harmala with tryptophane decarboxylase gene from C. roseus
have been shown to produce increased amounts of the secondary metabolites
nicotine and serotonin when expressing transgenes from yeast.
 Role of endophytes in in vitro production of secondary metabolites
 Bioreactors scaling up
 Immobilization scaling up of secondary metabolite accumulation
- Cell cultures of Plumbago rosea immobilized in calcium alginate and cultured in
MS basal medium containing 10 mM CaCl2 for the production of plumbagin
showed 3 fold increase as compared to the control.

3. By:
Mugdha Nigam
ME Biotech
2011H129007H

4. • Cancer is one of the deadliest diseases known through ages and has become
the leading cause of death worldwide in 2010.
• Cancer cells divide much faster than ‘normal’ cells. Therefore, two modes of
chemotherapy are possible:
- compounds that stop cell division
- cytotoxic compounds that destroy cells directly.
• The use of plant drugs for medical treatment is possible since plants have
evolved bioactive secondary metabolites (SM) that have been selected during
evolution as a defence system against, for example, microbes and herbivores.
• Due to their complex structure with several chiral centres important anticancer
agents are still extracted from plants and not synthesized chemically on a
commercial scale.

5.  Vinblastine and Vincristine
Source - Madagascar periwinkle, Catharanthus roseus
Parts used - pure alkaloids are extracted from aerial parts as important
medicines to treat breast, uterine cancer and Hodgkin’s as well as non-
Hodgkin’s lymphoma.
- Only organized shoot cultures have produced trace amounts of these dimeric
alkaloids.
 Camptothecin
Source - mainly obtained from the happy tree, Camptotheca
acuminata and related species (Cornaceae).
Parts used – Stem bark and young leaves
- The callus cultures grown on MS medium containing 4 mg/l
NAA, show accumulation of camptothecin upto 0.998 mg/l.
- Best results had been obtained with hairy root cultures of O.
pumila with a production of approximately 1mg/g dry wt of
CPT.

6.  P. hexandrum Royle or the Indian Podophyllum and P. peltatum L. or the American
Podophyllum of are only commercially exploitable plant sources of
Podopyllotoxin
 The rhizome and roots of P. hexandrum contains up to 6% of a resin podophyllin,
which is a rich source (up to 50%) of podophyllotoxin.

7.  Podophyllotoxin is a pharmaceutically active form of lignans.
It is used as a precursor for the synthesis of important antitumor drugs like
etoposide and teniposide , used in the treatment of lung cancer, testicular
cancer, a variety of leukemias and other solid tumours

8.  SIDE EFFECTS - Podophyllotoxin itself
has got severe gastrointestinal side effects
and is too toxic for therapeutic purposes.
 MODE OF ACTION - Inhibition of the
assembly of microtubules. Hence, cells
are arrested in the metaphase stage of
mitosis and cell growth stops.
The derivatives of podophyllotoxin are
topoisomerase II inhibitors arresting cells
in the late S or early G2 phase of the cell
cycle.
 PROBLEMS ASSOCIATED WITH
CONVENTIONAL PROPAGATION -
- P. hexandrum is an endangered species
in India.
- P. peltatum’s podophyllotoxin content in
the rhizome is much smaller than that in
P. hexandrum.
- Wild populations may be represented by
various genotypes growing under
different environmental conditions which
may affect drug profile leading to
problems in the purity of the final
product.

9.  Cell cultures of P. peltatum for production of podophyllotoxin was first attempted by
Kadkade et al.
 To increase the yield of podophyllotoxin, Woerdenberg et al. used a complex of a
precursor, coniferyl alcohol and b-cyclodextrin to P. hexandrum cell suspension
cultures. The addition of 3 mM coniferyl alcohol complex yielded 0.013%
podophyllotoxin on a dry weight basis, but the cultures without the precursor
produced only 0.0035%.
 Van Uden et al. initiated podophyllotoxin producing callus cultures from in vitro
plantlets of the Indian Podophyllum on B5 and MS media supplemented with growth
regulators, showing dark-grown cultures accumulated upto 0.3% podophyllotoxin
(dry weight basis).
 In vitro propagation of P. peltatum by using rhizome tips as explants has been
reported by Moraes-Cerdeira et al

10.  Suspension cultures from other species tried:
- Callitris drummondii
- Linum album
- L. nodiflorum
- L. mucronatum spp. armenum
 Anbazhagan et al. established embryogenic cell and adventitious root culture
systems in P. peltatum and revealed that adventitious roots contained higher
podophyllotoxin than embryogenic cell clumps through HPLC.

12.  Taxol is a complex diterpene alkaloid
found in the bark of the Pacific yew (Taxus
brevifolia Nutt.)
 Paclitaxel has been isolated from various
parts of other species of Taxus like T.
baccata, T. cuspidata, T. canadensis, T.
chinensis, T. x media, T. floridana, T.
yunannensis, T. mairei, T. sumatrana and
T. wallichiana.
 It is an antineoplastic agent, being widely
used majorly for the treatment of
advanced, progressive and drug refractory
ovarian cancer and breast cancer along
with the derivative taxases.

13.  It is also effective against noncancerous conditions like polycystic kidney
diseases and has shown promising results in multiple sclerosis and AIDS
related Karposi‟s sarcoma.
 MODE OF ACTION – Taxol promotes the polymerization of
microtubules but inhibits depolymerisation. This unusual stability blocks
the cell‟s ability to disassemble the mitotic spindle during cell division;
blocking it in the G2/M phase and eventually leading to cell death.
 PROBLEMS ASSOCIATED WITH CONVENTIONAL
PROPAGATION –
- T. Brevifolia is slow growing and is rarely found.
- The quantity of taxol obtained is in trace amounts (0.01% of dry weight
of the bark).

14.  Christen et al. reported the production of taxol by Taxus cell cultures.
 Fett-Neto et al. studied the effect of nutrients and other factors on paclitaxel production
by T. cuspidata cell cultures (0.02% yield on dry weight basis).
 Paclitaxel production by T. baccata cell suspension cultures was studied (1.5 mg/l).
 Maximum taxol production in T. cuspidata cultures obtained by adding Phenylalanine.
 Propagation of taxus seeds by embryo culture reported for T. brevifolia and other
species.
 Ketchum and Gibson showed addition of carbohydrate during the growth cycle
increased the production rate of paclitaxel in suspension cultures (14.5 mg/l).

15.  The production can be enhanced 17–19 fold by methyl jasmonate and
various other biotic and abiotic stress factors up to 35mg/l.
STRATEGIES FOR IMPROVING THE IN VITRO PRODUCTION
 Optimization of culture conditions
- Quantity and intensity of light affects Podophyllotoxin accumulation.
- Low oxygen content, temperature between 24-29 °C and high osmotic pressure
enhance taxol production.
 Optimization of culture medium
 Cell cloning and selection
 Cross species culture
- Coculture of Linum flavum hairy roots and P. hexandrum cell suspension
increased Podophyllotoxin accumulation.
- Cell suspensions of Taxus chinensis var. mairei cocultured with its endophytic
fungi, Fusarium mairei, showed 38-fold higher than that by uncoupled culture.
 Elicitation
- Salicyclic acid used as elicitor for podophyllotoxin.
- Methyl jasmonate used for eliciting taxol production.

16.  Large scale production
- Commercial production of podophllotoxin is yet to be achieved.
- Batch cultivation of T. baccata var. fastigata and T. wallichiana suspension
cultures has been carried out in 20 litre airlift bioreactor.
- 100-500 litre BTBBs have also been used for culture of T. cuspidata. After a
period of 27 days the yield of paclitaxel and total taxanes was 3mg l-1 and 74mg l-
1 respectively.
- Reactors with stirrers having low shear forces of 70,000 litre volume are used of
Taxol production (Phyton Biotech, Germany).

17.  Sustainable bioproduction of phytochemicals by plant in vitro
cultures: anticancer agents, Wink et al, 2009
 Studies on the production of some important secondary
metabolites from medicinal plants by plant tissue cultures,
Vanisree et al, Bot. bull Acad. Sin. 2004.
 Biotechnological approaches for the production of potential
anticancer leads Podophyllotoxin and Paclitaxel: An overview,
Anrini Majumder, Sumita Jha, eJournal of Biological Sciences,
2009.
 A review on trends in production of secondary metabolites
from higher plants by in vitro tissue, organ and cell cultures, S.
Karuppusamy, Journal of Medicinal Plants Research, 2009.