Current Research Journal of Biological Sciences 2(5): 345-351, 2010

ISSN: 2041-0778
© M axwell Scientific Organization, 2010
Submitted Date: March 10, 2010 Accepted Date: April 16, 2010 Published Date: September 15, 2010

Isolation, Characterization and Comparative Study on Podophyllotoxin

and Related Glycosides of Podophyllum heaxandrum
1
Phalisteen Sultan, 1 A.S. Shawl, 2 A.A. Abdellah and 3 P.W. Ramteke
1
Indian Institute of Integrative Medicine, Formerly, R RL -(CS IR), Sanatnagar,
Srinagar, 190 005 , Jammu and Kashm ir
2
Department of Biochemistry, Faculty of Medicine, AlBeida, 919- Libya
3
Departm ent of Biological Sciences, Allahab ad A gricultural Institute,
Deemed U niversity, A llahab ad, 2110 07, U ttar Pradesh , India

Abstract: HPLC, column and thin layer chromatography guided studies led to the isolation of seven different
compounds in methanolic extracts of Podophyllum hexandrum. The isolated compounds were analyzed using
L C-MS and High Performance Liquid Chromatography (HPLC ) studies interfaced to mass spectroscopy.
Isolated compounds were used successfully as chemical markers for the comparison of the twelve different
accessions of Podophyllum. We hav e also shown that the variation of chemical composition in P. hexandrum
agree well with their botanical phylogeny as revealed by genetic phylogeny. HPLC analysis also revealed
developm ent of valuab le chemoty pes containing hig her co ncen tration of isolated mark er com pounds.

Key w ords: Chemical, markers, podophyllotoxin, Podophyllum, spectroscopy

INTRODUCTION anticancer properties, podoph yllotoxin occu pies a very

Podophyllotoxin is a naturally occurring lignan,
which is extracted from the rhizomes o f Podophyllum
peltatum and P. hexandrum (Berberidaceae) and serves as
a starting compound for the prepara tion of the sem i-
synthetic cytostatics etoposide (VP-16-213) and
teniposide (VM-26) (Clark and Slevin, 1987; Holthuis,
1988; Stähe lin and Von W artburg , 1989 ). Pod ophyllum
hexandrum, a moisture and shade lov ing erect, glabrous,
succulent herb thriving from K ashm ir to Sikkim in
Himalayas at altitudes ranging from 2500-4000m (Fig. 1).
P. hexandrum has been extensively explo ited in
Ayurvedic system of medicine for treatm ent of ailments
like constipation, cold, biliary fever, septic wound s,
inflammation, burning sensation, mental disorder, genital
warts, mon ocytoid leukemia, Hodgkin’s and non
Hod gkin’s lymphoma (Singh and Shah, 1994). Extensive
chemical investigation of Podophyllum species revealed
presence of a number of compounds like podophyllin,
podophyllotoxin, querc etin, 4-demethylpodophyllotoxin,
podophyllotoxin gluco side, 4-dimethyl podophyllotoxin
glucoside, kaemp ferol, picro pod oph ylotox in,
deoxypod oph yllotox in, p icro p o d o p hy lotoxin ,
isopicropodophyllone, 4-demethyldeoxypodophyllotoxin,
"-peltatin and $- peltatin (S ingh a nd Shah, 1994 ).
Natural products have long been an important source
of treatments for cancer. Of the natural compounds with
important position (Imbert, 1998). Medicinal use of
Podophyllum hexandrum Royle (Himalayan Mayapple)
syn. P. emodi Wall (family, Berberidaceae) a high altitude
plant species native to alpine and sub-alpine regions of
Himalyas dates b ack to ancient times (Singh and Shah,
1994). The plant has been de scribed as A indri-a divine
drug in the Indian traditional system of medicine, the
ayurveda and has also been used in traditional Chinese
system of medicine (Wong et al., 2000) for the treatment
of number of ailments. In the modern allopathic system of
medicine, the plant has been successfully used for
treatment of various disorders, monocytoid leukemia,
hodgkins lympho ma, bacterial and viral infections
(Gowdey and C arpen ter, 1995; Co bb, 1990), veneral
warts (Beutner and Krog, 199 0), rheu matoid arthralgia
associated with n umb ness of the limbs and pyogenic
infection of skin tissues, AIDS assoc iated K aposis
sarcoma and different cancers of brain, lung and bladder
(Blasko and Cordell, 1998). Recently P. hexandrum
extracts have been found to offer radioprotection by
modulating free radical flux involving the role of lignans
presents (Chawla et al., 2006 ).
The roots and rhizomes of P. hexandrum are known
to synthesize a plethora of secondary metabolites besides
podophyllotoxin and related aryltetrahedronnapthalene
lignans with multifaceted pharmacological applications.
Etoposide and teniposide, the two semisynthetic glycoside

Corresponding Author: Phalisteen Sultan, Indian Institute of Integrative Medicine, Formerly, RRL-(CSIR), Sanatnagar, Srinagar,
190005, Jammu and Kashmir
345
 Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010
Fig. 1: Medicinal herb Podophyllum hexandrum in IIIM gene bank
derivatives form an integral part of the therapeu tic
regimen used for che motherapy and have triggered further
studies in the design and the synthesis of other potent
anticancer com pounds (C anel et al., 2000; Iseel, 1982;
Van et al., 1988). An analytical method for estimation and
characterization of the chemical constituents of this high
value medicinal plant is mandatory. Methods for
identification of aryltetrahydronapthalene and related
chemical marker lignans from P. hexandrum have rarely
been reported. Difficulty in obtaining the reference
standards is probably major reason for identification
of minor constituents. It is noted that
aryltetrahydronapthalene lignans occurring in nature are
all built around a common basic skelton and may show
under appropriate condition fragmentation pathway
ame nable to straightforward structural interpretation. It is
thus worth exploring the possibility of identifying these
constituents using their mass spec trome tric data with the
aim of registering a chem otaxo nom ic profile, w hich could
be diagn ostic value to herbs. The prese nt work reports the
use of LC-MS and HPL C for the identification of
important lignans from P. hexandrum.
MATERIALS AND METHODS
Ch em icals and reagents used: The major marker
compounds as described above were isolated in the
Natural Product Chem istry section of IIIM, Srinagar in
the year 2004 by rou tine chromatography techniques.
Identity and purity was confirmed by chromatographic
(TLC, HPLC) and spectral (IR, 1D- and 2D-NMR)
methods (Bastos et al., 1995). Solvents (water and
methanol,) were of HPLC grade and purchased from
Ranbaxy Fine Chemicals Limited (Okhla, New Delhi,
India). The structures were confirmed by their UV,
MS, 1 H NMR and 1 3 C NM R data compared with the
authe ntic data from literature. Acetonitrile of HPLC grade
(Aldrich, USA) and M illex syringe filter unit were
purchased from Reagent, New D elhi, India. Water for
preparation of samples and HPLC A D analysis was
346
deionized by a Milli-Q purification system with a 0.2 m
fiber filter (B arnstead, C A, U SA ).
Collection and identification of plan t ma terial:
Podophyllum plants growing in their natural habitat of
Himalayan Mountains were collected and transplanted
under partia l shade a t thre e diffe re nt gene banks of IIIM,
Srinagar at Bonera, Yarikha and Srinagar. The plant
material was identified by Department of Plant
Taxonomy, Kashmir University, Srinagar, India. The
voucher specimens of all the collected sam ples were
deposited at the H erbariu m of IIIM , Srinag ar, India.
Preparation of herbal extracts: The rhizomes samples
were taken from all the accessions grown at three
different location s viz. ge ne ba nk, IIIM -Srinagar, field
Station Bonera (Pulwama) and Yarikha (Gulmarg)- J and
K, India for preparation of phytoextacts. The dried plant
material was grounded to a fine powder and stored at 4ºC.
A known quantity of grounded sample was weighed and
subjected to sequential hot extraction using 100%
methanol. Contents were squeezed through muslin cloth,
and the filtrate from aqueous extract was filtered using
W hatman No.1 filter paper. The extraction process was
repeated three times (4 to 6 h). Organic solven ts were
clarified by centrifuga tion and then conc entrated to
dryness under reduced pressure. The known residue of the
extract was dissolved in HPLC prepa ratory tubes w ith
methanol (Dw ivedi et al., 1997).
Extraction and isolation of compoun ds: The dried and
pulverized roots of Podophyllum hexandrum (120 g) were
extracted with MeOH (300 mL) in a soxhlet over water
bath for 6 h. The extract was filtered and solvent was
removed in Rotavapor at 50ºC. The concentrated extract
was redissolved in HPLC grade methanol and volume was
adjusted to 1ml each. Injection volume 5uL and column
temperature 30ºC, flow rate 1m l/ minu te and wavelen gth
was set at 283 nm . The extracted portions were combined
and con centrated by evaporation under reduced pressure
 Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010
Fig. 2: HPLC chromatograms showing peaks of major marker compounds
to give a crude extract (18.5 g), which w as dissolved in
MeOH (100 m l).
HPLC analysis: Podophyllotoxin and its glycosides were
identified by HPLC based on the comparison of retention
time and UV spectrum with the reference compound.
HPLC analysis was performed on a ThermoFinnigan
HPLC mac hine w ith pum p syste m eq uippe d with a
966-photodiode-array detector, with the detection
wavelen gth set at 283 nm. Satisfactory separation was
obtained with reverse phase column utilizing a E. Merck
RP-18 column (250×4 mm, 5 :m) with a diode array
detector (SPDM-10 A VP/RF-10 AXL fluorescent
detector) and auto-injector STL-10 AD VP. Elution was
done with the mo bile phase (M eoH , H 2 O; 60:40) for 30
min. at a flow rate of 0.8 ml/min. A standardized mixture
of two marker compounds with known concentration of
podophyllotoxin and podophyllotoxin $-D glycoside were
used to create calibration curves (percentage area with
respect to the quantity of the pure compounds). Both the
marker com pounds exhibited eno ugh differences in th eir
retention times, which made their quantification easier.
LC-UV (DAD) chromatogram of the samples showed the
presence of two m arkers has been observed. The data was
statistically analysed for significant results.
347
Analytical HPLC cond itions: The chem ical analysis was
done on ThermoF innigan HPLC machine equipp ed w ith
auto sampler, column apartment and U V detector.
Acquiring and analysis of data was controlled by
Shemstation software (Agilent Tech, USA). A RP-18
column (0.3x150 mm) from E. merck was employed at
30ºC column temperature. Separation was done in the
isocratic mode using methanol and water (60:40) at a flow
rate of 0.8 ml/min with injection volume of 5 :L, UV
detection was set at 290nm. Prior to use, solvents w ere
filtered through a 0 .22 mm diameter m embrane filters.
Equal volum e of the standard solution was mixed and
injected in the HPLC system in volumes of 2, 4, 6, 8 and
10 :L for plotting calibration curves. Solutions w ere
injected in triplicate and the calibration curve s were
constructed by plotting value for concentration of each
analyte. Satisfactory separation was obtained as shown in
the chrom atograms (Fig. 2).
RESULTS AND DISCUSSION
The plant material was extracted with solvent system
methanol. Colum followed by thin layer chromatography
lead to the isolation of seven marker compounds. The
isolated compounds were labelled as pH-1, pH-2, pH-3,
 Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010

Podophyllotoxin 1-O--$ -D-glycoside Quercetin 3-O-$ -D-glycoside PPT, 1-O--$ -D-glycoside

Fig. 3: Various compounds isolated from phytoextracts of Podophyllum hexandum

Fig. 4: Fragmentation pathway of podophyllotoxin 1-0-$ -D glycoside

pH-4, pH-5, pH -6 and pH-7. After LC -MS analysis, the
identified compounds were found to be podophyllotoxin,
4 Demethyl podo- phyllotoxin, podophyllotoxin 1-0-"-D
glycoside, podophyllotoxin 1-0-$-D glycoside and
quercetin 3-0-$-D glycoside (Fig. 3) In the LC separation
it was found that gradient of methanol and water was the
optimal mobile phase.
Different marker compounds were identified by the
HPLC analysis in all the collected accessions.
Considerable variation in the chemical composition was

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 Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010

Table 1: H PLC analysis of different sam ples of Podophyllum hexandrum

Accession code R1 R2 R3 R4 R5 R1 R2 R3 R4 R5
PH 18-S 2.17 2.37 2.18 1.98 2.20 4.19 5.33 5.56 6.30 6.23
PH 18-S 1.91 2.09 2.06 1.95 1.89 7.49 6.33 5.34 6.53 5.21
PH 18-S 1.36 2.25 3.15 2.15 3.58 6.12 7.21 6.03 5.26 5.29
PH 18-S 2.35 4.00 2.96 3.44 1.95 5.21 5.51 4.98 6.11 5.59
PH 18-S 1.76 2.59 2.61 2.33 2.26 5.91 7.12 5.24 5.79 6.14
PH 18-S 1.66 2.96 2.03 3.41 2.35 5.72 4.68 5.82 3.99 5.09
PG r-S 2.06 1.99 1.89 2.23 1.68 4.50 3.10 3.02 4.10 3.78
PG r-S 2.06 2.11 2.17 2.03 1.98 2.09 3.02 2.51 1.84 2.44
PG r-S 2.21 1.19 2.72 1.76 2.07 2.64 2.19 3.10 1.86 2.70
PG r-S 2.89 2.21 2.32 2.97 2.06 2.87 2.47 3.25 3.51 5.05
PG r-S 2.40 2.78 2.39 1.99 2.19 2.81 4.70 1.99 3.92 2.28
PG r-S 2.61 2.47 2.29 2.00 1.98 4.25 4.61 5.03 2.98 3.23
PS-S 3.96 4.83 3.86 4.21 2.78 2.25 2.41 1.87 2.03 1.99
PS-S 4.79 5.96 5.02 4.02 5.16 2.06 1.95 2.07 2.35 1.47
PS-S 3.03 4.91 3.99 4.38 3.04 3.18 2.12 1.91 2.95 2.74
PS-S 5.31 3.42 4.23 5.65 3.19 1.91 3.18 1.86 2.74 2.36
PS-S 3.00 4.84 4.36 3.76 5.84 3.37 1.94 2.09 2.56 1.74
PS-S 4.29 4.33 3.98 4.49 4.36 1.87 2.61 0.78 1.31 0.83
Pp-B 2.20 2.68 2.93 2.88 3.21 6.68 5.36 4.12 3.18 6.13
Pp-B 3.19 4.11 3.17 3.51 3.32 5.91 3.33 5.09 4.41 3.86
Pp-B 2.52 3.72 2.55 3.47 2.59 2.53 5.44 5.45 4.99 4.84
Pp-B 3.09 3.24 4.11 2.45 3.31 4.61 4.84 5.41 5.23 4.91
Pp-B 2.79 3.09 3.11 4.21 3.85 5.49 5.25 4.48 4.60 6.23
Pp-B 3.12 4.09 3.78 2.68 4.30 4.92 5.33 5.22 4.27 5.21
PG -S 4.98 5.23 3.91 5.36 4.07 7.09 6.83 5.47 4.99 6.33
PG -S 4.12 5.13 4.76 5.78 4.06 5.41 6.03 5.68 5.91 4.92
PG -S 6.91 6.87 7.59 7.32 6.93 7.11 4.91 4.71 5.16 6.11
PG -S 5.21 4.91 6.12 5.03 5.18 6.49 5.12 5.41 6.19 7.39
PG -S 5.12 6.09 4.24 3.91 5.31 4.12 5.33 6.37 5.98 5.50
PG -S 5.75 6.29 6.33 4.92 5.11 4.92 5.31 4.97 5.71 5.81
PS-B 3.20 3.06 1.93 3.24 2.37 1.15 3.90 3.21 2.78 1.96
PS-B 4.98 2.07 3.84 2.95 3.41 2.00 2.51 2.41 2.91 1.68
PS-B 3.00 2.41 3.92 4.91 3.21 5.89 5.09 3.23 5.41 4.88
PS-B 3.55 5.92 3.28 5.84 4.41 2.08 3.70 4.28 5.52 4.22
PS-B 3.40 3.93 4.11 3.32 4.19 4.22 5.21 3.06 2.09 2.97
PS-B 3.55 3.41 3.92 3.65 3.22 6.3 3.50 5.60 4.20 3.86
PG -B 3.01 2.95 3.12 3.31 2.16 3.24 3.61 2.94 1.91 3.52
PG -B 2.33 3.93 3.01 3.97 3.01 2.54 2.21 2.81 2.78 3.01
PG -B 2.90 3.01 3.46 3.23 4.04 1.36 2.41 2.91 2.36 3.31
PG -B 3.06 2.23 4.22 3.07 2.22 0.79 3.47 2.68 4.09 4.22
PG -B 2.37 2.81 3.41 3.18 2.98 4.21 2.94 2.90 3.68 1.28
PG -B 3.53 4.02 1.26 4.51 3.33 3.91 2.68 1.36 4.91 3.49
PW -S 1.91 1.71 2.58 2.82 1.13 0.69 1.47 1.90 2.93 2.61
PW -S 2.21 1.47 1.63 2.06 2.38 2.79 0.92 0.91 -- 2.93
PW -S 2.56 2.18 1.87 2.34 1.30 1.10 2.07 1.85 1.66 2.43
PW -S 1.94 3.09 2.41 1.88 2.28 1.99 1.22 0.95 4.01 3.23
PW -S 2.11 2.09 2.91 2.78 2.36 2.80 1.98 2.11 3.39 4.22
PW -S 3.32 2.19 4.49 2.98 2.62 1.29 1.02 1.68 1.16 --
PV -S 1.81 1.92 2.09 2.07 2.01 0.86 2.65 2.68 2.41 2.00
PV -S 1.49 1.85 2.18 2.11 2.12 0.69 2.68 2.33 1.42 2.23
PV -S 2.02 2.81 3.09 3.17 2.66 2.41 1.34 1.01 0.92 0.90
PV -S 1.91 2.53 2.71 3.02 1.28 2.44 3.32 1.22 0.50 1.12
PV -S 2.41 2.81 3.12 2.01 2.90 2.40 2.18 2.03 1.86 0.92
PV -S 2.02 1.82 1.94 3.29 1.85 3.12 2.09 1.39 1.61 2.04
PY -S 1.57 1.84 2.09 2.26 2.14 3.29 2.18 1.89 2.11 3.68
PY -S 3.11 2.19 1.77 3.94 3.24 2.40 3.10 2.58 1.43 2.09
PY -S 1.12 0.98 2.13 2.53 1.99 1.08 2.33 1.85 2.71 0.93
PY -S 2.80 1.19 1.98 3.30 2.68 0.96 2.96 1.29 2.53 1.86
PY -S 2.41 3.03 2.71 1.71 2.39 2.11 0.99 4.02 3.68 2.45
PY -S 3.09 3.09 3.31 2.69 2.42 3.43 3.28 2.12 1.81 1.86
PY -B 1.91 2.38 4.11 3.68 2.83 2.95 3.13 2.37 3.74 1.91
PY -B 2.08 1.83 2.08 1.93 1.58 1.96 1.59 0.21 1.22 0.92
PY -B 1.55 2.60 2.59 1.93 1.58 2.92 1.41 0.92 3.46 0.84
PY -B 2.48 2.92 2.44 2.57 1.84 1.17 1.24 1.87 1.11 2.16
PY -B 2.64 2.79 2.37 2.24 3.21 0.94 0.41 3.21 2.91 2.68
PY -B 2.77 2.62 1.94 2.42 1.85 1.68 1.84 1.25 0.92 1.46
PSH -B 6.11 5.91 5.12 5.77 5.09 2.10 1.19 1.47 0.91 2.78
PSH -B 5.12 3.68 4.41 4.09 3.25 1.82 1.94 2.37 2.94 2.03
PSH -B 6.24 5.92 6.48 6.56 6.29 3.03 2.46 2.81 3.47 1.68
PSH -B 6.31 5.81 6.12 6.25 5.86 1.58 0.91 1.03 2.59 2.49
PSH -B 6.19 6.41 7.09 6.52 5.94 2.61 2.12 1.98 1.88 1.86
PSH -B 7.03 7.28 7.41 7.12 7.81 0.81 0.94 1.61 1.23 0.96

recorded in the analysed accessions as shown in Table 1. Podophyllum hexandrum samples obtained w ere
Using the experimental conditions reported above the a na lyze d. It w as found that the patte rns of their LC-MS

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 Curr. Res. J. Biol. Sci., 2(5): 345-351, 2010

Table 2: L ignans identified using LC-M S profiling of Podophyllum hexandrum

S a mp le na m e Retention time Percentage area of Mass (m/z) of R e te nt io n ti me Mass (m/z) of
(major peaks) major peak major peaks (minor peak) minor peaks
Stand ard P odo phyllo toxin 39.848 90.99 423 .1,43 7,39 7.1,3 13.1 30.495 313.1,246
Standard (4 Demethyl podophyllotoxin) 21.623 100 .0 423 ,383 .1, 28 8.9,1 85.1
PH -1 4 - D eme thyl po dop hylloto xin 21.634 80.75 423 ,383 .2, 24 6.5
PH-2 podophyllotoxin $-D-glycoside 25.882 100.00 599 .2,39 7.1,3 13.1
PH-3 podophyllotoxin " - D-glycoside 32.380 86.00 599.2,397.1,313 15.14 585 .2
413 .0
383 .0
217 .2
PH-4 Quercetin 3-0-$-D-glycoside 34.128 94.14 504 .2,32 5.1,2 99.1
PH-5 un identified 32.803 58.00 599 .2,39 7.1,2 29.0 39.756 599.2,
437,
397 .4
247 .0
PH-6 un identified 26.23 71.11 599 .2,39 7.1,3 13.0 32.67 599 .2
397.1,
365 .1, 21 7.0
PH-7 podophyllotoxin "-D-glycoside 39.82 98.00 437,397.2,313.0,247
184 .9

reconstructed chromatograms were more or less similar
except for som e variation in the relative intensity of
peaks. By studying, the fragmentation pattern as revealed
in corresponding LC-MS spectras, a number of
podophyllotoxin and related lignan marker compounds
were identified in various extracts (Fig. 4). The major
fragment ions observed in the mass spectra are
summarised in Table 2. The compound identification was
possible on the basis of the different fragmentation
pathways (Issel et al., 1998; Rahman et al., 1995 ; Clark
and Slevin, 1987). To ensure that observed (M+Na) ion
fragm ents of glycosylated lignans was indeed the same as
observed in previous spectras. Due to the high sensitivity
of instrument, this method was particularly advantageous
for the sam ples of limited quantity. Usin g this
methodology, detailed structural information was obtained
for lignans, lignan glycosides and other second ary
metabolites. HPLC analysis prov ides a re producible
retention time using stand ardised conditions for
development of lignan database. UV spectra collected on
line provided evidence for general classification and sub
structures of each comp ound.
DISCUSSION
The prime objective of the stud y was to inv estigate
the chem otaxo nom ic studies of different P. heaxandrum
extracts. W ith this distribution p attern, popdophyllotoxin
and its glycosides are potential taxonomic markers
although they apparently. Pod ophyllum has been
exten sively investigated due to its use in Ayurvedic and
Chinese medicine. Roots of P. heaxandrum have given a
number of glycosides. Plant samples from different
regions were analyzed and the occurrence of the
glyco sidic compounds was confirmed by a combined set
of criteria including retention time, mass spectra of both
positive and negative mode. All major peaks that could be
recognized as glycosides w ere taken into consideration.
The results were summarized and compared with the
previous reports for isolation and spectral identification of
iridoids from the studied species. Due to diversity of
species and h abitats, the results may be much more
complex. Therefore, it seems that more attention should
be paid to the scrutiny of various toxic ingredients rather
than the determination of a few kinds of compounds or
only one with high sensitivity. This conclusion is also
supported by the following data from the herbal
preparations. The HPLC technique appeared particularly
useful for screening the UV absorbing glycosides in
P. heaxandrum lead to the detection of these compounds.
Detailed metabolite p rofiles for selected com pounds in
these P. heaxandrum samples demo nstrated that the
chemical compositions of these accessions was infact
quantitatively different because these plants were not
grown under identical conditions at the same time or in
the sam e location.
ACKNOWLEDGMENT
Autho rs are highly thankful to Dire ctor, IIIM,
Srinagar for financial support of the project. Autho rs are
highly thankful to Miss Safeera of GLC section for
computer help in drawing chemical structures. Also like
to thank to the contribution of Maxwell Scientific
Organization for providing extensive su pport financially
to publish it online.
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