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324 Biotechnology and Chemistry of Podophyllum
active substance in Podophyllum but was subsequently shown to be inactive. He also
isolated podophyllinic acid and quercetin. The chemical analysis of P. emodi began with
investigations based on materials from the roots and it was shown that the components of
the two different Podophyllum species were the same. It was identified along with its
epimer picropodophyllotoxin. Various chemical investigations have shown that the resin
consisted mainly of podophyllotoxin, deoxypodophyllotoxin, 4'-demethylpodophyllotoxin
and - and -peltatins. The presence of quercetin, pigments and their glucoside derivatives
were also reported.
A microanalytical technique for determination of podophyllotoxin in P. hexandrum
roots was developed by Mishra et al. (2005a) quantitatively through RP–HPLC and RP–
HPTLC. Podophyllotoxin in the extract ranged from 4.25 to 5.22% and 4.15 to 5.12% by
HPLC and HPTLC respectively. The podophyllotoxin content in resin was estimated as
4.91–5.57% and 4.90–5.58% by HPLC and HPTLC respectively.
A RP–HPTLC method was also developed for the quantification of podophyllotoxin
in P. hexandrum, collected from different locations of the western Himalaya of India,
ranging from 3250 to 4100 m above the mean sea level (Mishra et al., 2005b). Seven pairs
of diastereoisomers of podophyllum lignans at the C4 position, including three pairs of
spin-labelled compounds, have been separated within 20 min (Slaninaa and Glatz, 2004).
The topical application of podophyllin in Condyloma acuminatum produced very
satisfactory clinical results and podophyllin caused pronounced cytological changes in
normal human and rabbit skin. These reports initiated a renewed medicinal interest in
podophyllin from the standpoint of its antimitotic activity. Podophyllin is still the drug of
choice in the treatment of Condyloma acuminatum. The continued study of podophyllin
based upon its recognition as a cytotoxic agent and the growing knowledge of the nature
and biological properties of pure constituents involving a multitude of chemical and
biological investigations have been conducted over a period of more than a century. Initial
expectations regarding the clinical utility of podophyllotoxin were tempered largely due to
its unacceptable gastrointestinal toxicity. Using special procedures to inhibit enzymatic
degradation, these researchers indeed obtained the podophyllotoxin--D-glucopyranoside
as the main component and its 4'-demethyl derivative from the Indian Podophyllum species.
The research efforts were focused on to chemically modify podophyllotoxin, which
eventually led to the discovery of clinically important anticancer drugs etoposide and
teniposide (Fig 20.3).
During the drug developmental stages, it began to become clear that etoposide
induced double stranded breaks by stabilizing a complex formed between topoisomerase II
and DNA that is referred to as the cleavable complex. Topoisomerase II was the most likely
cellular target in the double stranded breaking activity of epipodophyllotoxin like etoposide
and teniposide. The cytotoxicity of analogues of etoposide was associated with inhibiting
DNA topoisomerase II by stabilizing the covalent topo II- DNA cleavable complex.
Although etoposide is active in the treatment of many cancers and is widely used in the
therapy, it presents several limitations, such as moderate potency, poor water solubility,
development of drug resistance, metabolic inactivation, and toxic effects. Therefore in
order to obtain better therapeutic agents and extensive synthetic efforts have been devoted
to overcome these problems.
With increasing information about its structure–activity relationships, wide
investigations have generated exciting chemotherapeutic candidates and successful
applications of drug development from podophyllotoxin-related lead, such as etopophos,
NK-611, GL-331, azatoxin, TOP-53, tafluposide.
Biotechnology and Chemistry of Podophyllum 325
O
O
O
O
O
OCH
3
OR
H
3
CO
O
O
R
1
HO
R
2
NH
O
O
O
O
OCH
3
OH
H
3
CO
R
R R
1
R
2
R
H CH
3
OH
Etoposide NO
2
GL
331
H
S
OH Teniposide F NPF
PO(OH)
2
CH
3
OH Etopophos
H CH
3
N(CH
3
)
2
.HCl.H
2
O NK611
O
O
O
O
OCH
3
OH
H
3
CO
N
N
O
O
OCH
3
OH
H
3
CO
N
H
TOP-53 Azatoxin
Fig. 20.3. Chemical structures of podophyllotoxin derivatives.
Etopophos
The problem of the low solubility of etoposide was resolved by introducing a phosphate
group at position 4': this led to the selection of etopophos which was better suited for
intravenous administration. In fact, the highly soluble compound was shown to be a
prodrug for etoposide itself. The phosphate group rapidly hydrolyzed by phosphatases
contained in the plasma, thereby generating etoposide in the circulatory system (Schacter et
326 Biotechnology and Chemistry of Podophyllum
al., 1994; Budman, 1996). This prodrug was developed and many of the side effects of
other, more or less toxic analogues of etoposide were eliminated.
NK-611
In NK-611, the alcohol group at position 2" of the ethylidene glucoside species is replaced
with a dimethylamino group. Introducing an amine both renders the new compound
soluble, because of the generation of its hydrochloride, and improves its bioavailability as a
result of the molecule-altered physicochemical properties. The antitumour activity of this
compound against human tumour xerograft seemed to be promising and was shown to be
similar to that of etoposide; however it proved to be a more potent topoisomersae II
inhibitor and was more cytotoxic against a variety of human cancer lines including lung,
gastrointestinal, ovarian, testicular, breast, head and neck tumour cells and leukaemia.
GL-331
GL-331 exhibits cytotoxicity, topoisomerase II inhibitory activity and potency against
tumours. This new epipodophyllotoxin derivative doesn’t have the glucoside group which
was believed to be necessary for activity, but rather had a p-nitroanilino group present at 4-
position. Structural studies at this position involving replacement with aniline or an amine
(Lee et al., 1989, 1990) showed that compounds containing an aniline group at 4- position
had a higher level of inhibitory activity for human topoisomerase II. Various structural
modifications made at this position showed that there was no correlation between human
topoisomerase and DNA breaking associated with the protein. GL-331 showed topo II
inhibition and caused DNA double-strand breakage and G2 phase arrest, it could induce
cell death by stimulating protein tyrosine phosphatase activity and apoptotic DNA
formation. GL-331 was also shown to be active in many multidrug-resistant cancer cell
lines. It is known to show good stability, biocompatibility and its pharmacokinetic profiles
are similar to those of etoposide.
Azatoxin
Azatoxin only differs from podophyllotoxin with respect to: (i) replacement of the
benzodioxole group with an indole ring; (ii) the presence of a nitrogen atom at the
asymmetrical centre at C and (iii) the presence of a phenol at position 4', essential for the
anti-topoisomerase I1 activity. Molecular modelling had shown that only an indole ring at
that position gave a tight fit with podophyllotoxin. Nevertheless, the cleavage induced by
azatoxin differed from that induced by other topoisomerase II inhibitors like VP-16,
amsacrine or mitoxantrone. Although it was almost as potent as VP-16 in inhibiting
topoisomerase II, it differed in the fact that it also inhibited the polymerization of tubulin at
low concentrations.
Biotechnology and Chemistry of Podophyllum 327
TOP-53
Structural studies of podophyllotoxin analogues based on molecular modelling techniques
resulted in the rational design of a range of new chemical agents. It was almost as potent as
etoposide in inhibiting topoisomerase II. TOP-53 was then selected for evaluation from a
series of 4-alkyl amino derivatives of 4'-O-demethyl-4-desoxypodophyllotoxin
synthesized. TOP-53 displayed twice the inhibitory activity of etoposide against
topoisomerase II and exhibits in vivo superior antitumour activity than etoposide against
different types of cancer. In view of high activity and good properties, TOP-53 has been
progressed to phase II clinical trials (Terada et al., 1993).
Tafluposide
More recently, 2",3"-bis pentafluorophenoxyacetyl-4',6'-ethylidene--D-glucoside of 4'-
phosphate-4'-demethylepipodophyllotoxin has been obtained. It is a novel catalytic
inhibitor of topoisomerase I and II and has shown outstanding antitumour activity in vivo
against a panel of experimental human tumour xenografts. Tafluposide showed significant
cytotoxicity against cells derived from either haematological or solid tumours, with a
marked inter-patient variation (Sargent et al., 2003).
Extensive modifications have been done in the four rings (A, B, C, D) of
podophyllotoxin to assess the effects of different structural moieties. Diverse
norbornenecarboxylate esters of podophyllotoxin and its epimers and diastereoisomers have
been prepared through Diels–Alder cycloaddition by treating the dienophilic acrylates of
cyclolignans with cyclopentadiene (Fig. 20.4).
Pharmacological Action
It has been shown that the mechanism of cytostatic action at the cellular level of
podophyllin was the same as that of colchicine, i.e. an inhibition of the formation of the
mitotic spindle, resulting in an arrest of the cell division process in metaphase and a
clumping of the chromosomes (c-mitosis). Later it was shown that, at the subcellular level,
this is due to binding of podophyllotoxin to tubulin, preventing these macromolecules to
form microtubules, which constitute the fibres of the mitotic spindle. In contrast to
podophyllotoxin, neither etoposide nor teniposide has any effect on tubulin assembly. It has
been reported that etoposide and teniposide are not inhibitors of microtubule due to the
presence of the bulky glucoside moiety, and suggested that they must be inhibiting cell
proliferation by some other mechanism. It was concluded that these drugs arrested cells in
either the late S or early G2 phase of the cell cycle and they detected single-stranded breaks
in DNA by the interaction of drug with DNA in HeLa cells. Further research indicated that
etoposide does not interact with DNA in vitro to cause cleavage but rather induces single
stranded breaks in DNA only in situ.
328 Biotechnology and Chemistry of Podophyllum
H
N
O
O
O
O
OCH
3
OCH
3
H
3
CO
N
O
O
OH
O
O
OCH
3
OCH
3
H
3
CO
N
O
O
O
O
OCH
3
OCH
3
H
3
CO
R
H
O
O
O
O
OCH
3
OCH
3
H
3
CO
O
R
O
O
O
O
O
Fig. 20.4. Structures of synthetic analogues of podophyllotoxin.
Apart from antitumour and antiviral activity, insecticidal and phytotoxic activities of
podphyllotoxin are also reported. Initially, it was hypothesized that podophyllotoxin exerts
its pharmacological effect by producing potent spasm in blood vessels with resultant
ischaemia, necrosis and sloughing. The anti-mitotic effects of podophyllotoxin are the
result of the drug’s ability to act as an inhibitor of microtubule assembly. Podophyllotoxin
also appears to attach to cell proteins and acts by increasing the incorporation of amino
acids into proteins, inhibition of purine synthesis and inhibition of purine incorporation into
RNA. It has also been found to have a direct effect on mitochondria by reducing the activity
of cytochrome oxidase and succinoxidase. The mode of their antiviral activity is by tubulin
binding, reverse transcriptase inhibition, integrase inhibition and topoisomerase inhibition.
Podophyllotoxin is most notable among the tubulin binding lignans. By binding to tubulin,
these are able to disrupt the cellular cytoskeleton and interfere with some critical viral
processes.
Biosynthesis of Podophyllotoxin and Analogue Compounds
The full biosynthetic route of cyclolignans has not been elucidated yet, but several studies
in different species of Forsythia, Linun and Podophyllum led to the proposition of a
pathway (Petersen and Alfermann, 2001). In the initial steps, coniferyl alcohol was
synthesized from phenylalanine (Fig. 20.5) by phenylpropanoid enzymes (Van Uden et al.,
1990). Instead of an alternative route to give the polymeric product lignin, lignans are
obtained by dimerization of coniferyl alcohol to yield pinoresinol (Davin et al., 1997).
After several steps, this compound is transformed into matairesinol and in multistep
Biotechnology and Chemistry of Podophyllum 329
reaction the intermediate yatein yields deoxypodophyllotoxin (Bromhead et al., 1991; Xia
et al., 2001). Several feeding experiments were carried out in cell cultures to elucidate the
biosynthetic relationships among the natural cyclolignans (Van Uden et al., 1995). In
Linum flavum cell cultures, 6-methoxypodophyllotoxin was obtained as the major
cyclolignan and this compound was not obtained when the cell cultures were fed with
podophyllotoxin. Otherwise, 6-methoxypodophyllotoxin was obtained when
deoxypodophyllotoxin or -peltatin were added to the cell cultures. In P. emodi
podophyllotoxin and its -D-glucoside were the cyclolignans obtained when the cell
cultures were fed with deoxypodophyllotoxin. The enzymes deoxypodophyllotoxin-7-
hydroxylase and deoxypodophyllotoxin-6-hydroxylase, both isolated from cell cultures of
Linum spp. (Petersen and Alfermann, 2001; Molog et al., 2001), are the proteins that carry
out the hydroxylation on positions 7 or 6 of deoxypodophyllotoxin to give either
podophyllotoxin or -peltatin, respectively.
COOH
NH
2
CH
2
OH
OMe
OH
O
O
OMe
OH
HO
OMe
H
H
O
OMe
OH
HO
OMe
H
H
HO
OH
OH
OH
MeO
HO
MeO
OH
MeO
HO
MeO
O
O
OMe
MeO
O
O
O
O
OMe
O
O
O
O
OMe
OMe
MeO
Lignan
Lignans
Pinoresinol
Phenyl alanine
Coniferyl alcohol
Matairesinol
Lariciresinol
Secoisolariciresinol
Yatein
Deoxypodophyllotoxin
Fig. 20.5. Proposed biosynthesis of cyclolignans related to podophyllotoxin.
330 Biotechnology and Chemistry of Podophyllum
Developments in the Biotechnological Aspects
Because of the limited supply of Podophyllum rhizomes, due to their intensive collection in
the wild, there is considerable interest in both defining and biotechnologically exploiting
the podophyllotoxin biosynthetic pathway, thereby making this compound more generally
available (Lewis and Davin, 1999).
Cell cultures have been used in the past for the production of natural products in vitro
as an alternative. The production of podophyllotoxin from callus cultures has been reported
for P. peltatum (Kadkade, 1981, 1982). It is very difficult to establish callus cultures in P.
hexandrum. Van Uden et al. (1989) reported initiation of cell cultures from the roots of P.
hexandrum and production of podophyllotoxin in undifferentiated callus and cell
suspension cultures. It has also been reported that coniferin was a substrate for
podophyllotoxin production (Van Uden et al., 1990). Van Uden et al. (1989) tested
hundreds of basal medium, hormone combinations and concentrations and found B5
medium supplemented with 2% coconut milk, 4% sucrose, 4 mg/l naphthalene acetic acid
to be the best. The cell suspension cultures of P. hexandrum do not appear to secrete
podophyllotoxin in the culture medium as the amount of podophyllotoxin in the medium
was reported to be negligible. Culture conditions and other complex factors are involved in
the biosynthesis of podophyllotoxin. Woerdenbag et al. (1990) reported increased
podophyllotoxin content in P. hexandrum cell cultures after feeding coniferyl alcohol
complexed with -cyclodextrin. Cells cultured in media containing 4 mg l
1
NAA
accumulated 0.001–0.002% podophyllotoxin on dry weight basis. A new route for
secondary metabolite production is the use of natural vector system.
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. A variety of fungal products can elicit inducible
defensive plant responses in both host and nonhost plants and that such responses can also
be triggered by plant products released during cell-wall degradation. Physical damage to the
leaves of P. hexandrum resulted in the accumulation of podophyllotoxin glucosides and
aglucone at the wound site (Oliva et al., 2002).
Since podophyllotoxin has been recognized as a starting compound for the chemical
synthesis of etoposide (VP-16-213) and teniposide (VM-26), its ever increasing demand
has led to the investigations on the biotechnological production of podophyllotoxin
(Petersen and Alfermann, 2001; Puri et al., 2006). Submerged cultivation of P. hexandrum
cells for podophyllotoxin production was studied in a 3l stirred tank bioreactor fitted with a
low-shear Setric impeller (Chattopadhyay et al., 2002). Batch cultivation of P. hexandrum
was conducted in a 3l stirred-tank bioreactor using statistically optimized process
parameters. The plants exhibit delayed emergence of functional leaves or hypocotyl
dormancy. On GA
3
treatment the functional leaves (Khushwaha et al., 2007) were found to
emerge at a favourable temperature of 25ºC in a higher percentage of seedlings and in a
shorter time. Functional leaves emerged even at 10ºC, a temperature when hypocotyl
dormancy generally prevails. For further growth and development in all the seedling parts
there is a considerable increase in the biochemical parameters related to carbon and
nitrogen metabolism, respiration and total dehydrogenase activity also indicated an
enhancement of metabolic processes as influenced by GA
3
.
Puri et al., (2006) isolated an endophytic fungus Trametes hirsuta, which has been
shown to synthesize podophyllotoxin and other aryltetralin compounds in vitro. Culture
conditions for growth and productivity were also standardized by these workers. The
Biotechnology and Chemistry of Podophyllum 331
availability of a biotechnological route for production of aryltetralin lignans is a step
forward in the direction of gaining independence from the endangered plant species.
Kharkwal et al. (2002) have developed a method for inducing improved seed
germination in a high altitude P. hexandrum species through hot water treatment at 40–
100ºC for 30–120 seconds. Plants of P. hexandrum, collected from lower, mid, and upper
distribution limits in alpine Himalaya were studied under greenhouse conditions to evaluate
the photosynthetic response (Vats and Kumar, 2006). The maximum net photosynthetic
rates (P
N
) and stomatal conductance (g
s
) were measured in the considered population
during the 3–6th week of development.
Etoposide is derived chemically from podophyllotoxin by two step conversion. It has
been demonstrated that a cell line of P. peltatum active in the biosynthesis of
podophyllotoxin was able to maintain repeated biotransformation by oxidative coupling of
the butanolide to the podophyllotoxin analogue. 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 (Franssen and Walton, 1999).
Conclusion
Lignans are believed to act as defense compounds in the producing plants. For mankind, the
lignans play a role as health-promoting agents in dietary plants on the one hand and in
cancer treatment on the other. Although P. hexandrum has been considered a rare and
threatened species, large scale removal (generally during the middle of growing season and
well before seed set in the wild) of its underground parts still continues at rates well over
natural regeneration. Therefore, special attention needs to be given for its propagation and
conservation. It must be emphasized that greater awareness needs to be created and the
local inhabitants should be motivated to cultivate medicinal plants as alternative (high
value) crops, including trials at lower altitudes.
Acknowledgements
Authors are grateful to the Director, IHBT (CSIR), Palampur for providing required
facilities. Financial assistance to Abha Chaudhary during the course of the project (NWP-
0037) from CSIR, New Delhi, India is gratefully acknowledged. This is IHBT
Communication No. 1002.
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