Seasonal variation in content of camptothecin from the bark of Nothapodytes nimmoniana (Grah.) Mabb., using HPLC analysis

Abstract

To study and compare seasonal variation in camptothecin (CPT) content from bark samples of Nothapodytes nimmoniana obtained from geographically and climatologically isolated populations.
A standard High Performance Liquid Chromatography methodology was used to analyze and quantify CPT from bark samples of N. nimmoniana.
Sample collected from Amboli yielded highest CPT content 1.337 g/100 g dry bark powder during the monsoon compared to other localities in study. Monsoon (August) showed to accumulate higher levels of CPT in barks of N. nimmoniana as compared to summer (May). Amboli averaged highest accumulation of CPT compared to other localities under study.
These findings indicate season to have control over accumulation of CPT. Locality Amboli has highest CPT content in all seasons and were the elite population during the study. The study also suggests the need for further investigation in lights of biosynthesis in the plant.

Full text

Pharmacognosy Research | July-September 2013 | Vol 5 | Issue 3 219
Address for correspondence:
Dr. Sandeep R. Pai, Department of Plant Tissue Culture,
Regional Medical Research Centre, ICMR, Nehru Nagar,
Belgaum-590 001, Karnataka, Maharashtra, India
E-mail: drpaisr@gmail.com
Seasonal variation in content of camptothecin from the
bark of Nothapodytes nimmoniana (Grah.) Mabb., using
HPLC analysis
Sandeep R. Pai1,4, Nilesh V. Pawar2,4, Mansingraj S. Nimbalkar3,4, Parthraj R. Kshirsagar3, Firdose K. Kolar3,
Ghansham B. Dixit3
1Department of Plant Tissue Culture, Regional Medical Research Centre, ICMR, Belgaum, Karnataka,
2Department of Botany, The New College, Shivaji Peth, 3Department of Botany, Laboratory of Cytogenetics and Plant Breeding, Shivaji University,
4Department of Research and Development, Sahyadri–Group Enriching, Nature, Environment and Science, Kolhapur, Maharashtra, India
Submitted: 11-02-2013 Revised: 21-02-2013 Published: 22-05-2013
ORIGINAL ARTICLE
INTRODUCTION
Forests are imperative in regulation of the water cycle
and stabilizing soils. They also help in soaking carbon
dioxide and balancing oxygen levels. In addition, forest
provides habitat for diverse ora and fauna, offer cultural,
spiritual, and recreational opportunities, and provide a
variety of food, medicines, and wood. The forest cover of
India is 19.27 % of the geographic area, corresponding to
63.3 million hectare.[1]
Over 50 % of all modern clinical drugs are of natural
product origin.[2] Plant secondary metabolism is a paradigm
for the metabolic diversity found in nature. Several tree
species from Western Ghats is gaining importance due
to its newly discovered pharmaceutical and curative
properties. Nothapodytes nimmoniana (Grah.) Mabb.,
(Icacinaceae), (Syn.: Nothapodytes foetida, Mappia foetida)
is one such plant. It is a rich source of potent alkaloid
camptothecin (CPT) and 9-methoxycamptothecin.[3-5] The
metabolites extracted from N. nimmoniana show anti human
immunodeciency virus, anti-neoplastic, and anti-malarial
activity.[3] Many researchers have suggested High Pressure
Liquid Chromatography to be one of the best analytical
methods to detect and quantify plant metabolites.[6-9] CPT
(mol. Formula C20H16N2O4 and mol. wt. 348.4) is an
alkaloid originally isolated from a Chinese tree Camptotheca
acuminata (Nyssaceae).[10] In this context, there has been
an enormous demand world-wide for the alkaloid CPT.
Herein, we present work on the effect of the season on
content of CPT accumulation.
MATERIALS AND METHODS
Sampling and sample processing
Field surveys were carried out in the Southern parts of
Maharashtra State (MS), India (part of Western Ghats)
PHCOG RES.
Objective: To study and compare seasonal variation in camptothecin (CPT) content from
bark samples of Nothapodytes nimmoniana obtained from geographically and climatologically
isolated populations. Methods: A standard High Performance Liquid Chromatography
methodology was used to analyze and quantify CPT from bark samples of N. nimmoniana.
Results: Sample collected from Amboli yielded highest CPT content 1.337 g/100 g dry
bark powder during the monsoon compared to other localities in study. Monsoon (August)
showed to accumulate higher levels of CPT in barks of N. nimmoniana as compared to
summer (May). Amboli averaged highest accumulation of CPT compared to other localities
under study. Conclusion: These ndings indicate season to have control over accumulation
of CPT. Locality Amboli has highest CPT content in all seasons and were the elite population
during the study. The study also suggests the need for further investigation in lights of
biosynthesis in the plant.
Key words: Camptothecin, High Performance Liquid Chromatography,
Nothapodytes nimmoniana, seasonal variation
ABSTRACT
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DOI: 10.4103/0974-8490.112434
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Pai, et al.: Seasonal variation in camptothecin content
220 Pharmacognosy Research | July-September 2013 | Vol 5 | Issue 3
for locating populations of N. nimmoniana. Voucher
specimen was deposited in the herbarium, Laboratory of
Angiosperm Taxonomy, Department of Botany, Shivaji
University, Kolhapur (MS) India (voch. no. SRP-NN 1/05).
Western Ghats is one of the global biodiversity hot spots
with discrete tropical climate and distinct forest types.
Like other parts of India, Southern Maharashtra receives
three seasons viz. monsoon, winter and summer. The
peak periods being in the months of August, December
and May selected as representatives for the three seasons.
Five sites (Amba, Amboli, Chandoli, Naikwadi, and
Panhala) were sampled with different geographical and
cliamtological settings. Plants above 15 cm gbh were
selected from each population. Samples collected were
oven dried at 50 ± 2°C for 48 h and powdered. The
powdered samples were stored in plastic containers in
cool dry place until use.
Extraction
The samples were prepared by the method employed by Pai
et al.; Fulzele and Satdive[5,11] with following modications.
The samples for HPLC analysis were prepared by exposing
1 g of dry bark powder suspended in 15 ml 90% aqueous
methanol to microwave extraction technique. The extracts
were filtered through filter paper and centrifuged at
10,000 × g for 10 min. The volumes of yielded supernatants
were adjusted to 15 ml with the same solvent. The extracts
were re-ltered through a 0.2 µ nylon lter and were used
for the HPLC analysis.
Quantitation of CPT using HPLC
Instrumentation
The HPLC analysis was performed on Waters
chromatographic system (Model no. 2690) consisting
of a quaternary pump, manual injector, and UV
detector. The Waters software system was used for
HPLC data processing. Chromatographic separation
was achieved on a Waters C 8 column (Symmetry, 5 µm,
4.6 mm× 250 mm).
Chromatographic conditions
Mobile phase consisting of A (acetonitrile) and B (water)
was used for separation with 40 % A as to 60% B in an
isocratic mode with injection volume 10 µL. The ow
rate was 1.6 mL/min and the detection wavelength of the
dual λ absorbance detector beam was set at 254 nm and
240 nm. The analysis time was 10 min for both, standards,
and samples used for the analysis.
System suitability
The system suitability test was assessed by three replicate
injections of the standard solutions at a particular
concentration. The peak areas of which were used to
evaluate repeatability of the proposed method and their
peaks were analyzed for resolution.
Calculations, calibration curves and linearity
CPT was accurately weighed and dissolved in few drops of
Dimethyl sulfoxide (DMSO) by warming and the volume
was made accordingly with methanol to produce a standard
stock solution (mg/mL). The stock prepared was warmed
to dissolve CPT completely and avoid turbidity. The stock
solution was then serially diluted with methanol to prepare
the working solutions for the calibration curves at ve
concentration levels (25, 250, 500, 750, and 1000 µg/mL).
All the solutions were stored in microfuge tubes at 4°C.
RESULTS
The selected sites have fairly rich distribution of the
plant population, dense patch of individuals were seen
at Chandoli, Amboli, and Naikwadi where as localities
Amba and Panhala had scattered patches. Flowering was
observed from August to October. Fruit setting continues
until December, where during later part of the month
fruit ripens. Poaching marks in the form of cut trees were
observed at the localities Chandoli and Amboli, indicating
arguably high-risk of these plants to be vanished from
the areas in the near future. The other localities were well
preserved and still untouched.
Quantitation of CPT using HPLC
HPLC analysis of different concentrations of
standard CPT yielded proles with a retention time of
5.389 (±0.3) min. Sharp and clear peaks of standards were
obtained. A linear calibration curve for the CPT within the
concentration range of 25-1000 µg (R2= curve coefcient
0.981) was obtained by plotting concentration of CPT
against Area Under Curve for respective concentration
peak. The calibration equation (y= 26437x+ 2E+ 06)
obtained for the curve was used to calculate the CPT
concentration.
Fifteen samples were chemically proled for understanding
seasonal effect on CPT content [Figure 1]. Proles for
the samples collected from 5 localities during 3 seasons
were evaluated and compared with the retention time of
standard CPT. Validation of the method was carried out
by spiking 100 µl (750 µg) of standard CPT to 100 µl of
the extract of bark collected during the summer (May)
from Naikwadi and the recovery was within the range of
95-100%.
CPT content during monsoon (August)
The peaks obtained from HPLC analysis were sharp enough
to identify CPT content [Figures 2a and e]. The proles also
show other peaks clustering at different retention times.
Sample collected from Amboli yielded highest (1.337 g/100
g dry bark powder) CPT content followed by Naikwadi,
Amba and Panhala [Figure 1]. Sample from Chandoli yielded

Pai, et al.: Seasonal variation in camptothecin content
Pharmacognosy Research | July-September 2013 | Vol 5 | Issue 3 221
very low amount (0.252 g/100 g dry bark powder) of CPT as
compared to other localities within this season.
CPT content during winter (December)
The peaks of CPT in the HPLC prole obtained by
injecting the extracts of the bark samples obtained from
different localities during the winter (December) are
presented in [Figure 2f and j]. A range from 0.490 to
1.490 % CPT accumulation in bark samples collected in
the season from the localities under study. Highest percent
accumulation of CPT was observed in samples collected
from Amboli (1.490 %) during the season, followed by the
samples collected from Amba > Naikwadi > Chandoli >
Panhala [Figure 1].
CPT content during the summer (May)
The peaks obtained for the HPLC analysis carried out for
the extract of the samples obtained during the summer
(May) were sharp and isolated [Figure 2k and o]. Bark
sample collected from the locality Amboli showed highest
percent accumulation of CPT in the season, followed by
samples collected from Amba > Naikwadi > Chandoli >
Panhala [Figure 1]. The amount was comparatively less than
that recorded during the monsoon (August) and winter
(December) for all the localities except Chandoli.
Correlation for the content of CPT and the altitudinal
differences of the sites of the collection had no signicance.
Further, it was seen that the correlation coefcient during
each season was varying widely with P values > 0.05 level
(August: R= 0.578, P= 0.307), (December: R=−0.243,
P= 0.6939), (May: R= −0.4781, P= 0.4153). The results
were in accordance with Padmanabha et al.[12] wherein they
have revealed that the difference in content of CPT yield
among different sites could not be attributed to either
geographical location (latitude) or altitudinal differences.
More likely, environmental (seasonal) and genetical
background of the population are the regulating factors.
Seasonal variation in CPT content
Seasonally, monsoon (August) showed a rise in the yield
of CPT content in all localities tested except Amboli and
Chandoli where winter (December) demonstrated high
yield [Figure 2]. Difference between highest and lowest
content of CPT for each of the locality was more than 50%.
Locality Chandoli, Amba, and Panhala showed 55.94, 55.34,
and 51.06 % difference respectively between highest- lowest
content of CPT. Similarly, difference between highest
to lowest content of CPT during the respective seasons
among all the localities were: August: 81.15 % difference
between Amboli (1.337 %) and Chandoli (0.252 %),
December: 66.91 % difference between Amboli (1.490 %)
and Panhala (0.493 %), May: 52.93 % difference between
Amboli (0.786 %) and Panhala (0.370 %).
DISCUSSION
The content of CPT quantied by HPLC method illustrated a
pattern where monsoon averaged highest followed by winter
and summer (August > December > May). Quantitation
of 15 extracts of dried, powdered bark material gave
values from 0.252to 1.490 % of CPT [Figure 2]. The
lowest content of percent CPT was lower than that
reported (0.4%) by Ramesha et al .[13] Over all production of
CPT was highest in all seasons in locality Amboli, whereas
other localities in order from high to low content of
% CPT after Amboli are Amba, Naikwadi, Panhala, and
Chandoli.
Thus, it is fascinating to know the underlying reasons for
high production of CPT. Though, content of CPT has
been less studied from genetical point of view, we make
here an attempt to know the seasonal (environmental)
effect on yield of CPT. Seasons have shown to play a
key role in regulation of many physiological processes,
and ultimately rate of metabolism.[14] It is well-known
that the secondary metabolites are inuenced, either
qualitatively or quantitatively, by the age of the plant,
variety, and climatic conditions.[15,16] The probability of
difference due to developmental stages in the plant under
study was minimized by selecting mature trees over
16 gbh. Seasonal changes are quantitative and qualitative,
but the possibility cannot be entirely ruled out that
particular biochemical pathways can be switched on or off
as a result of environmental inuence.[17]
CPT with all other terpinoid indole alkaloid’s (TIAs), are
derived from the common precursor strictosidine, which
is the product of a condensation reaction between indole
tryptamine and terpenoid secologanin.[18] Tryptophan
(Trp) biosynthesis is proved to be essential in primary
and secondary metabolism in C. acuminata another CPT
yielding plant. The biosynthesis is governed by enzyme
Trp synthase, which is made of two subunits (A and B).
The abundance of Tryptophan synthase subunit B (TSB)
Figure 1: Content of camptothecin (%) from bark extracts of
N. nimmoniana collected from various localities during different seasons

Pai, et al.: Seasonal variation in camptothecin content
222 Pharmacognosy Research | July-September 2013 | Vol 5 | Issue 3
messanger Ribo Nucleic Acid (mRNA) and protein were
paralleled to production of CPT in C. acuminata and was most
abundant in vascular tissues, especially, cambium, primary
xylem, and primary phloem.[19] TSB is highly expressed in
C. acuminata during early seedling developmental stages
corresponding to accumulation of CPT, consistent with
the idea that Trp biosynthesis (primary) and secondary TIAs
pathway are coordinately regulated.
Trp once formed is converted to tryptamine which plays a
vital role in TIAs biosynthesis by acting a bridge between
primary and secondary metabolism. The enzyme required
for this conversion is Tryptophan decarboxylase (TDC).
The enzyme is extensively studied in Catharanthus roseus
and it is well-understood that it is encoded by a single copy
gene[20] and its elicitation occurs at transcriptional level.[19]
Whereas, Lopez-Meyer and Nessler[21] have showed that
Figure 2: High Pressure Liquid Chromatography (HPLC) proles of samples collected during (a-e): August; (f-j): December; (k-o): May and where
(a, f, k): Amba; (b, g, l): Amboli; (c, h, m): Chandoli; (d, i, n): Naikwadi; (e, j, o): Panhala are localities
a
b
c
d
ejo
i n
h m
g l
fk

Pai, et al.: Seasonal variation in camptothecin content
Pharmacognosy Research | July-September 2013 | Vol 5 | Issue 3 223
TDC in C. acuminata is coded by 2 genes(TDC1 and TDC2)
and out of which TDC1 is developmentally regulated and
TDC2 serves as part of defense mechanism. TDC1 had
highest expression in apex, young stem and bark, which
also contains the highest level of CPT. Biosynthesis of
TIAs has been well studied in C. roseus,[22] C. acuminate,[23] and
Ophiorrhiza pumila,[24] which indicate a level of complexity
to the multicellular nature of TIAs biosynthesis. It has
also been proposed that the intermediates of TIAs are
translocated from interior sources to epidermis and
ultimately to the sink (laticifers and idioblasts).
This serves a rationale to our study, where we nd monsoon
(August) to accumulate higher levels of CPT in barks of
N. nimmoniana as compared to summer (May). The plant
under study is deciduous in nature and pre-monsoon
showers elicit the sprouting and blossoming of the tree,
which progresses to elevate primary metabolism. Previous
work on TIAs and CPT biosynthesis signify primary
metabolism to be directly linked to secondary metabolism,
which in turn is related to TIA and CPT biosynthesis.
However, more detailed studies are required for explaining
apparent discrepancy in biosynthesis and accumulation.
ACKNOWLEDGMENTS
Authors are indebted to Head, Department of Botany, Shviaji
University, Kolhapur. SRP is thankful to Ofcer-in-charge,
RMRC, ICMR, Belgaum and Indian Council of Medical
Research, New Delhi for support.
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Cite this article as: Pai SR, Pawar NV, Nimbalkar MS, Kshirsagar PR, Kolar
FK, Dixit GB. Seasonal variation in content of camptothecin from the bark of
Nothapodytes nimmoniana (Grah.) Mabb., using HPLC analysis. Phcog Res
2013;5:219-21.
Source of Support: Nil, Conict of Interest: No.