International Journal of Applied, Physical and

Bio-Chemistry Research (IJAPBCR)

ISSN(P): 2277-4793; ISSN(E): 2319-4448
Vol. 5, Issue 3, Dec 2015, 1-8
TJPRC Pvt. Ltd.

IDENTIFICATION AND QUANTIFICATION OF PODOPHYLLOTOXIN FROM

PODOPHYLLUM HEXANDRUM BY ESI-LC/MS/MS
SHARMA D.K & KUMAR A
Department of Chemistry Himachal Pradesh University, Shimla, Himachal Pradesh, India

ABSTRACT
An ESI-LC/MS/MS method for the quantification of podophyllotoxin from Podophyllum hexandrum
population from Sangla valley of Kinnaur, Himachal Pradesh, India in the Northern Himalayas was developed.
Qualification of podophyllotoxin was performed by thin layer chromatography (TLC) with Rf values of 0.85 (leaf) and
0.94 (root) when compared with the standard podophyllotoxin. UV-VIS spectrophotometeric studies showed electronic
absorption at max value 284 nm. Gradient chromatographic separation of Podophyllotoxin was performed on a YMC-Pak
Pro C-18 : 4.6mm 250 mm, HPLC column, packed with 1.5 m particles equipped with a 0.7m pre-filter (Supleco),
using mobile phase methanol/water (1/1, v/v) and 100% methanol, both containing 0.1% ammonium hydroxide (25%)
and 10 m mol ammonium acetate (pH 9). Structural information from the tandem mass spectrometric data compared with

could be employed in the absence of reference standards for the marker and was particularly useful in view of the scarcity
of the chemical standard of podophyllotoxin.
KEYWORDS: Podophyllotoxin; Podophyllum Hexandrun; ESI-LC/MS/MS; Tandem Mass Spectrometry

Received: Oct 05, 2015; Accepted: Oct 12, 2015; Published: Oct 18, 2015; Paper Id.: IJAPBCRDEC20151

INTRODUCTION
Podophyllum hexandrum also known as Indian Mayapple, which is an endangered medicinal perennial
herb is belonging to the family Berberidaceae. Podophyllum hexandrum, a moisture and shade loving erect,
glabrous, succulent herb thriving from Himalayan zone at an altitude between 1300 to 4300 m above from sea level.
Extensive chemical investigation of Podophyllum species revealed presence of a number of compounds like
podophyllin, podophyllotoxin, querectin, 4-dimethypodophyllotoxin, kaempherol, picropodophyllotoxin, -peltatin
and -peltatin [1]. The podophyllotoxin content in Indian Podophyllum is more (7-15%) in comparison to other
species notably Podophyllum peltatum (4-8%), the most common species in American sub-continent [2]. Recently
podophyllotoxin has acquired great importance and high medicinal status due to its effectiveness as antimitotic,
anticancer and immunostimulatory activity [3,4,5] especially for curing uterine tumors [6]. The rhizome powder of
the plant is used as laxative or to get rid of intestinal worms and also used as poultice to treat warts and tumorous
growth on the skin. Semi synthetic derivatives of podophyllotoxin e.g. etoposide (VP-16) [7], etopophos [8] and
tenoposide (VM-26) are effective against the treatment of lung cancer, a variety of leukaemia, and other solid
tumours [9].
Recently, Podophyllum hexandrum extracts have been reported to offer radioprotection by modulating free
radical flux involving the role of lignans presents [10-16]. Due to its anticancerous property podophyllotoxin is in
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Original Article

those of lignin markers already reported for the herb established the quantitative high yield of pophyllotoxin. The method

Sharma D.K & Kumar A

increased demand throughout the world. Total synthesis of podophyllotoxin is an excessive process and availability of
compound from natural resource is an important issue for pharmaceutical companies that manufacture these drugs [17].
The annual supply is at present estimated as 50-80 tonnes, while the demand is more than 100 tonnes. To meet this ever
increasing demand of crude drug, the rhizomes of Podophyllum hexandrum are being indiscriminately collected in large
quantities [18]. As, a result Podophyllum hexandrum is reported as endangered species in Himalayan region. Traditionally
developed methods with TLC, HP-TLC and HPLC for podophyllotoxin quantification from Podophyllum hexandrum are
longer, time consuming and costly leading to higher material cost and increased energy consumption, labour and even
hazardous for nature with use of several non-green solvent systems [19-20]. In the present work we report a new, simple,
reproducible and cheap ESI-LC/MS/MS method for the identification and quantification of podophyllotoxin from
Podophyllum hexandrum roots and leaves from Sangla Valley, Kinnaur, Himachal Pradesh,India.

MATERIAL AND METHODS

Chemicals and Solutions
Acetonitrile (HPLC grade), HPLC grade water (Ranbaxy, Mumbai), Chloroform, n-Hexane, Ethyl acetate,
Ethanol, Methanol (all Merck), 25% NH4OH, 0.1% HCOOH, 10mM NH4OAC all Analytical grade prepared in HPLC
grade water. Podophyllotoxin standards were purchased from Sigma-Aldrich chemicals USA and were at least of 99%
purity.
Sample Collection and Preparation
Plant materials (Roots and leaves of podophyllum hexandrum) for our research study were collected from the
Sangla valley (3300m altitude) of Kinnaur, Himachal Pradesh, Northern Himalayas during the months of October (2014),
January and May (2015). Plants specimens were identified and confirmed at Department of Forest Products, University of
Horticulture and Forestry, Nauni, Solan, H.P. India. Plant tissues (Roots and leaves) of Podophyllum hexandrum
(separately, one by one) were pulverized under liquid nitrogen after air drying in shades to get their powder. One gram of
each sample powder was put in 10 ml of falcon tube with methanol, vortexed for 10 minutes. Kept the tubes as such
overnight, centrifuged at 3000 rpm for 20 minutes at room temperature and then collected the methanolic supernatant after
filtering the extract, ready for further qualitative and quantitative analysis.
Qualitative TLC Analysis
To perform thin layer chromatography analysis of leaf and root tissues of Podophyllum hexandrum, above
methanolic supernatant mixture was used for TLC spotting in triplicate. Chloroform and methanol (9:1) solvent system
was standardized for the TLC analysis to calculate Rf value of root and leaf extracts comparing with standard
podophyllotoxin spot
HPLC Analysis
Podophyllotoxin analysis was performed by High performance liquid chromatography (HPLC). The HPLC
equipment consisted of a 1200 series binary pump (G1312B), a 1200 series gradient pump (G1310A) and a degasser
(G1379B) (Agilent, Technologies, Germany) connected to an autosampler with chemstation 6.0 version software package.
Gradient chromatographic separation of Podophyllotoxin was performed on a YMC-Pak Pro C-18: 4.6mm 150 mm,
HPLC column, packed with 2.5 m particles equipped with a 0.7m pre-filter (Supleco). The injection volume was 10L
and the column oven temperature was set to 30C. Mobile phase A was methanol/water (1/1, v/v), mobile phase B was
Impact Factor (JCC): 1.9028

Index Copernicus Value(ICV): 3.0

Identification and Quantification of Podophyllotoxin from Podophyllum Hexandrum by ESI-LC/MS/MS

100% methanol, both containing 0.1% ammonium hydroxide (25%) and 10 mmol/L ammonium acetate (pH 9). A gradient
elution was performed with 100% A for 0.5 min, a linear increase to 50% A until 25 min, followed by 0% A from 30 until
40 min and re-equilibration from 41 to 45 min with 100% A. The flow rate was optimized and set to 0.5ml/min.

Figure 1: HPLC Chromatogram for the Podophyllotoxin Standard Material

Figure 2: HPLC Chromatogram for the Podophyllum Hexandrum Root Sample

Figure 3: HPLC Chromatogram for the Podophyllum Hexandrum Leaf Sample

Analysis of Podophyllotoxin by ESI-LC/MS/MS
LC-MS/MS was done on tripple quadruple, 3Q-ESI-EMD/MS and API 4000 Q-Trap MS/MS instrument. To
achieve a fast separation we chose a reversed-phase C18 column with a small particle diameter of 1.5 m. Similar to most
previous methods a watermethanol gradient was used for LC separation. A hybrid triple quadrupole linear ion trap mass
spectrometer API 4000 Q-Trap equipped with a Turbo V source ion spray operating in positive ESI mode was used for
detection (Applied Biosystems, Darmstadt, Germany). High purity nitrogen was produced by a nitrogen generator NGM

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Sharma D.K & Kumar A

22-LC/MS (cmc Instruments, Eschborn, Germany). To minimize contamination of the mass spectrometer, the column flow
was directed only from 1.0 to 5.0 min into the mass spectrometer using a diverter valve. Otherwise methanol with a flow
rate of 2.50L/min was delivered into the mass spectrometer. The Turbo Ion Spray source was operated in the positive ion
mode using the following settings: ion spray voltage =4500 V, ion source heater temperature = 500 oC, source gas 1 = 45
psi, source gas 2 = 40 psi and curtain gas setting = 25 psi. Analytes were monitored in the multiple reaction monitoring
(MRM) mode, mass transitions and MS parameters. Quadrupoles Q1 and Q3 were working at unit resolution.

Figure 4: ESI-3Q-EMD Mass Spectra for the Podophyllum Hexandrum Leaf Sample

Figure.5: ESI-3Q-EMD Mass Spectra for the Podophyllum Hexandrum Root Sample
Matrix Effects
Due to the use of crude samples of podophyllum hexandrum and the fast LC separation, we analyzed the influence
of the matrix on signal intensities. Solvent systems and buffers like methanol, water, 0.1% ammonium acetate and 25%
ammonium hydroxide shows a variation in the quantification of podophyllotoxin from Podophyllum hexandrum. The
presence of Podophyllum hexandrum extract caused a mean signal reduction (S.D.) of 205%, calculated with the
Chemstation 6.0 version software, 1200 series Agilent Technologies

Impact Factor (JCC): 1.9028

Index Copernicus Value(ICV): 3.0

Identification and Quantification of Podophyllotoxin from Podophyllum Hexandrum by ESI-LC/MS/MS

OH

OH
O

O
O

MeO

MeO

OMe

OMe
OH

OMe

Dimethylpodophyllotoxin

Podophyllotoxin

OH

OH

OH

O
HO

HO

HO

HO

HO

HO
OH

O
OH

HO

Quercetin 3-O--D-glycoside
MeO

OMe
OMe

PPT, 1-O--D-glycoside
Figure 6: Various Compounds Present in the Phytoextracts of Podophyllum hexandrum
The major fragment ions masses observed in the mass spectra are summarised in Table 1. The compound
identification was possible on the basis of the different fragmentation pathways. Since the molecular mass for
podophyllotoxin is 414.405, so we found ESI-MS of podophyllotoxin in the leaf as 415.2 as [M+1] + peak and 415.2 [M+1]
+

in the root.
Table 1: HPLC & Mass Spectrometric data for Podophyllotoxin
Retention Time (R.T)
(minutes)
28.4
28.5
28.3

Sample
Standard
Leaf
Root

HPLC Content %age

100
2.01
4.28

Mass (m/z) ratio

[M]+ or[M+1]+
414.405
415.2
415.2

Table 2: Summary of Validation Parameters of RP-HPLC Method

Sr.No.
1
2
3
4
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Parameters
Linearity and Range g/ml
Correlation coefficient
Accuracy (% Recovery)
Precision (% RSD)*
Intra- Day

Experimental Value
10-80
0.9997
99.92-100.14
0.250
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Sharma D.K & Kumar A

5
6

Inter- Day
Ruggedness(% RSD)*
Robustness(% RSD)*
7
Change in Wavelength
8
Change in Flow Rate
9
LODa g/ml
10
LOQb g/ml
*All the values expressed as a mean Six Determination
a
LOD = Limit of detection.
b
LOQ =Limit of quantitation.

0.328
0.516
0.015
0.031
0.68
1.05

RESULTS AND DISCUSSIONS

It was concluded that podophyllotoxin content is present in higher amount in plant population collected from
Sangla valley region of Kinnaur in Himachal Pradesh, higher altitude Himalayas. . Chromatographic content of
podophyllotoxin was found to be 2.01% podophyllotoxin in leaf and 4.28% in root sample of Podophyllum hexandrum.
These plant specimens collected from the higher altitude range (3300m) of Himachal Pradesh, therefore it was found that,
and content of podophyllotoxin was less in leaf compared to root. As the altitude increased and this phenomenon is
reversed in case of leaves of P. hexandrum. The applied HPLC method is specific and can be referred for the simultaneous
analysis of other active constituents in P. hexandrum plant and its products with good sensitivity, precision, and
repeatability.
By studying, the fragmentation pattern as revealed in corresponding mass spectra, a number of podophyllotoxin
and related lignan marker compounds were identified in various extracts. To ensure that observed [M+Na] ion fragments of
glycosylated lignans was indeed the same as observed in previously reported spectras. Due to the high sensitivity of
instrument, this method was particularly advantageous for the samples of limited quantity. Using this methodology,
detailed structural information was obtained for lignans, lignan glycosides and other secondary metabolites. HPLC analysis
providfes a reproducible retention time using standardised conditions for development of lignan database. UV spectra
collected on line provided evidence for general classification and sub structures of each compound.
A single laboratory validation (SLV) method was developed and validated as per ICH guidelines with RP-HPLC
and LC-MS/MS technique. Here we present a simple, short, reproducible and low cost ESI-LC/MS/MS method for the
determination of podophyllotoxin from Podophyllum hexandrum plant samples. Here, we report 2.01% podophyllotoxin in
leaf and 4.28% in root sample of Podophyllum hexandrum and consequently molecular mass 415.2 [M+1] + for leaf and
415.2 [M+1] + for root sample using ESI- 3Q-MS analyser.

CONCLUSIONS
An ESI-LC/MS/MS method for the Identification and quantification of podophyllotoxin from Podophyllum
hexandrum was developed based on LC/MS/MS finger printing of the standard marker. The method could be employed in
the absence of reference standards for the marker and was particularly useful in view of the scarcity of the chemical
standard of podophyllotoxin. It is believed that the same analytical strategy can be applied to the quantification of other
medicinal herbs, especially where reference standard markers are difficult to obtain.

ACKNOWLEDGEMENTS
Authors are thankful to Sprint Testing Solutions Ltd, Mumbai, India for providing necessary analytical support.

Impact Factor (JCC): 1.9028

Index Copernicus Value(ICV): 3.0

Identification and Quantification of Podophyllotoxin from Podophyllum Hexandrum by ESI-LC/MS/MS

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