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Simultaneous Spectrofluorimetric Determination of Scopoletin and

Mangiferin in a Methanolic Extract of Canscora decussata Schult.

Article  in  Asian Journal of Traditional Medicines · October 2008

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 Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional
Medicines, 2008, 3 ( 6 )

Regular Articles

Simultaneous spectrofluorimetric determination of

scopoletin and mangiferin in a methanolic extract of
Canscora decussata Schult.
Neeraj K. Sethiya a, Alok Nahata a, V. K. Dixit a*
a. Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya
Sagar (M.P.) 470003, India

Abstract

Scopoletin and mangiferin are two phenolic compounds detected in Canscora decussata (Gentianaceae). A simple, accurate and
sensitive method for their determination in crude drug was developed using spectrofluorimetry. Scopoletin exhibited a strong bright
blue fluorescence at excitation and emission wavelengths of 430 nm and 460 nm, respectively, and mangiferin exhibited a strong
apricot yellow green fluorescence at excitation and emission wavelengths of 248 nm and 520 nm, respectively. The method was
statistically validated and found suitable for quantitation of mangiferin and scopoletin in the methanolic extract of C. decussata. The
proposed spectrofluorimetric method provides a faster and low cost quality control by simultaneous routine analysis of scopoletin
and mangiferin in C. decussata and its formulations.

Key words: Canscora decussata; gentianaceae; spectrofluorimetry; coumarin; xanthone; scopoletin; mangiferin; shankhpushpi

Introduction Ayurvedic Pharmacopoeia of India, Shankhpushpi

I n t h e Ay u r v e d i c s y s t e m o f m e d i c i n e ,
‘Shankhpushpi’ is considered as ‘Medhya Rasayana’
–meaning a drug, which rejuvenates, maintains,
and potentiates intellect and memory. Formulations
containing ‘shankhpushpi’ as a principal constituent
are widely advertised in Indian print and electronic
media as memory enhancers. According to the
* Author to whom correspondence should be addressed. Prof.
V.K. Dixit Address:Department of Pharmaceutical Sciences, Dr.
Hari Singh Gour Vishwavidyalaya, Sagar (MP) 470003. India; Tel.:
+91-7582-264582; Fax: +91-7582-264163; E-mail: dixitvk2011@
rediffmail.com
Neeraj K. Sethiya E-mail: nscognosy2006@gmail.com
Alok Nahata E-mail: aloknahata@gmail.com
Received: 2008-05-27 Accepted: 2008-10-20
consists of the whole plant of Convolvulus pluricaulis
Choisy (Convulvulaceae). The pharmacopoeial
monograph also mentions in its note that Clitorea
ternatea Linn. (Papilionaceae) and Evolvulus
alsinoides Linn. (Convulvulaceae) are used as
Shankhpushpi in certain parts of India [1]. In the
eastern parts of India, Canscora decussata Schult.
(CD) (Gentianaceae) is popularly known as
“Shankhpushpi” and found up to an altitude of 1300
meters [2]. It is also grown in Sri Lanka and Myanmar.
The entire plant, as well as its fresh juice, is used
in medicine. It is used in the treatment of insanity,
epilepsy and nervous debility. This plant contains
bitter substances, oleoresin, triterpenes, alkaloids [3-5]
and xanthones such as mangiferin [6, 7]. The presence
of scopoletin in CD has been detected for the first time

224
 Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional
Medicines, 2008, 3 ( 6 )
by a co-TLC method.
Fluorescence under UV light is a characteristic
of the majority of natural coumarins [8-10]. These
compounds are very easily detected, since they give
characteristic fluorescent colors under UV light, which
are intensified by further treatment with ammonia
vapors. Scopoletin is a coumarin, which exhibits
a blue violet fluorescence under UV light [11, 12].
Mangiferin and Scopoletin both contain heterocyclic
fused rings, which are responsible for their intense
fluorescence under UV light.
Mangiferin and scopoletin in methanolic
solution exhibit a bright blue fluorescence and a light
green fluorescence, respectively, under UV light.
Therefore, it was thought worthwhile to develop
a sensitive, specific, simple, precise and accurate
spectrofluorimetric method for the estimation of
mangiferin and scopoletin in the methanolic extract of
CD.
Materials and methods
General experimental procedures
The spectrofluorimetric study was carried out
with a Shimadzu RF 5301 PC spectrofluorimeter, to
determine the levels of fluorescence of the phenolic
compounds in a stationary state. The light source
used was a xenon 150 W lamp with an optical system
composed of two automatic monochromators, one
for excitation and the other for emission, of a mesh
type to enable a suitable wide selection of excitation
and emission wavelengths for the coumarins [13]. A
quartz cell was used [14]. The detection system was
an R 450-01 photomultiplier which transformed the
fluorescent radiation emitted by the scopoletin solution
in the cell into an electrical signal. Scopoletin (99.98
%) was obtained as a gift from Laila Impex Research
Centre, Vijaywada (A.P.), India and pure mangiferin
(99.96 %) was obtained from Natural Remedies Pvt.
Ltd., Bangalore, India.
Plant material
225
Aerial parts of CD were collected from Raipur
district, Chattisgarh, India, from January to March,
2007. The herb was identified by Prof. S.C. Agarwal,
Head, Department of Botany, Central Drug Research
Institute, Lucknow and preserved in the herbarium
of the institute (Voucher Specimen no. NS/ AN/ Cd-
2409).
Preparation of extracts
Aerial parts of CD were shade dried at room
temperature. Extraction was performed according to
the method described by Nahata et al. [15]. The shade
dried plant material (500 g) was coarsely powdered
and subjected to extraction with petroleum ether
(1500 ml) in a Soxhlet apparatus. The marc was
then extracted with methanol (1500 ml) to obtain
the methanolic extract. The yield of the extract was
found to be 3.45 % (w/w). All chemicals used for the
procedure were of analytical grade.
Isolation of scopoletin
The presence of scopoletin in CD was detected
for the first time by co-TLC method with a standard.
It was then isolated by column chromatography
with silica gel G (80-120 mesh) and its identity
was confirmed by its melting point (204 °C), UV
absorption maxima and superimposable FTIR spectral
analysis. Additional NMR evidence further confirmed
the identity of the isolate as scopoletin.
Interpretation of spectral data of the isolate
The FTIR spectrum of the isolated compound
is characteristic of 6-methoxy, 7-hydroxy coumarin
i.e. scopoletin. The FTIR spectrum was identical
to the reference standard which proved this. The
H NMR spectrum showed a three proton singlet at
3.9δ corresponding to -OCH3, two pairs of doublets
centered at 6.1 δ and 7.7 δ corresponding to C 3-H
and C4-H with J=9 cps indicating that these protons
were on adjacent carbon atoms of a double bond, two
aromatic protons (singlets) C8-H and C5-H at 6.8 δ and
6.9 δ respectively, stretching at 9.9 δ indicative of a –
 Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional
Medicines, 2008, 3 ( 6 )
OH which shows one exchangeable proton with D2O.
After taking into consideration the melting point and
the analytical data obtained from UV, IR and NMR
analysis, the isolated compound was considered to be
scopoletin.
Preliminary analysis
A preliminary analysis was carried out to
determine the wavelength at which maximum intensity
is exhibited by pure scopoletin and mangiferin. For
this purpose, 100 µg/ml samples of pure scopoletin
and mangiferin were prepared in methanol. They
were scanned spectrofluorimetrically to obtain the
excitation and emission wavelengths. The λ max
shown by scopoletin had an excitation at 430 nm
and an emission at 460 nm while the λ max shown
by mangiferin had an excitation at 248 nm and an
emission at 520 nm.
Preliminary spectrofluorimetric screening of the
methanolic extract for testing the wavelength
range
The methanolic extract of CD was scanned in the
spectrofluorimeter in concentrations of 0.1 µg/ml and
0.01 µg/ml. The scanning was performed at excitation
and emission wavelengths of 200 nm to 700 nm and
absorption maxima was found at 315 nm and 435 nm.
This wavelength corresponded to λmax of mangiferin
and scopoletin, respectively. No other absorption
peaks were detected in the range screened. Scanning
between 248 nm and 520 nm did not exhibit any other
peak, thus the range selected for analysis was expected
to screen only mangiferin and scopoletin in the test
sample.
Preparation of standard curve
Standard curves of scopoletin and mangiferin
were prepared in methanol. First of all, stock solution
containing 100 µg/ml of scopoletin and mangiferin
was prepared in methanol. Then, this stock solution
was used to prepare required dilutions containing
5, 10, 15, 20, 25 and 30 µg/ml of scopoletin and
226
mangiferin. The volume of stock solution used was
0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml, respectively. The
samples were analyzed in the spectrofluorimeter
against solvent blank (methanol). The wavelength and
intensity of each sample was recorded and a standard
curve was prepared for concentration versus the
intensity of fluorescence.
Determination of scopoletin and mangiferin
concentration in methanolic extract
10 mg of the methanolic extract was weighed
accurately and dissolved in 10 ml of methanol with
vigorous shaking. It was then filtered and the volume
made up to 100 ml with methanol. This solution was
analyzed in the spectrofluorimeter and intensity of
fluorescence was recorded. The concentration of both
scopoletin and mangiferin in the extract samples was
determined from their standard curves.
Spectrofluorimetric method development for
simultaneous estimation of scopoletin and
mangiferin
After the success of the spectrofluorimetric
analysis in determining the concentration of scopoletin
and mangiferin individually in the methanolic extract
of CD, it was thought worthwhile to develop a method
for the simultaneous estimation of scopoletin and
mangiferin concentrations in crude drug samples.
For this purpose, 1 g shade dried powdered drug was
taken and subjected to methanolic extraction. The
methanolic extract was transferred to a volumetric
flask and the volume made up to 100 ml with
methanol. The fluorescence intensity of this diluted
sample was determined by spectrofluorimetry. The
whole procedure was repeated three times to obtain
triplicate readings. The concentration of scopoletin
and mangiferin in all the samples was obtained by
extrapolating from the standard curves. The mean
concentration of scopoletin and mangiferin present
in 1 g crude drug was determined. After determining
the concentration of scopoletin and mangiferin per
ml of the methanolic extract, the mean concentration
 Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional
Medicines, 2008, 3 ( 6 )
per gram of crude drug (mg/g) was calculated. The
scopoletin and mangiferin content in crude drug
powder of CD was found to be 4 mg/g and 7 mg/g,
respectively.
Thus, a simple spectrofluorimetric method for
analysis of scopoletin and mangiferin in CD was
developed. Further recovery studies were performed
to validate this novel analytical method.
Analytical method validation
- Linearity
Standard solutions (5 µg/ml to 30 µg/ml) were
prepared in methanol and the intensity of fluorescence
was recorded in the spectrofluorimeter. The standard
curve was prepared by plotting the concentration
as the abscissa versus the intensity of fluorescence
as the ordinate. A linear dependence of intensity
on concentration was observed over the entire
concentration range tested.
- Precision and accuracy
The precision of the method was checked
using standard solutions of the methanolic extract at a
concentration of 0.1, 0.2, 0.5 and 1.0 µg/ml, prepared by
appropriate dilutions with methanol. The solutions
were analyzed in a spectrofluorimeter at 430 nm and 460
nm (excitation and emission wavelengths) for scopoletin
and 248 nm and 520 nm (excitation and emission
wavelengths) for mangiferin and the intensities were
recorded. The corresponding concentrations were
extrapolated from the standard curve.
The entire procedure was repeated three times for
each dilution and the readings were expressed as Mean
± S.D. (n=3). Then, 0.1 µg/ml solutions of scopoletin
and mangiferin were prepared by appropriate
dilutions and analyzed spectrofluorimetrically. The
concentrations of scopoletin and mangiferin were
calculated for the sample.
This sample of known concentration was
added in an equal volume (1 ml) to all the
previous dilutions, and analyzed to see whether
227
the observed concentrations corresponded to the
theoretical concentrations from the standard curve.
The % recoveries were calculated on the basis
of determination of the analyte added to a sample
containing a known amount of scopoletin and mangiferin
(Table 1).
Results and discussion
Standard curves for scopoletin and mangiferin
were prepared at excitation and emission wavelengths
of 430 nm and 460 nm and 248 nm and 520 nm,
respectively, using a spectrofluorimeter. In both cases,
the plots of concentration versus intensity exhibited a
linear relationship. The equation of the straight line
for scopoletin was y=34.642x and that for mangiferin
was y=5.157x-114.06. A methanolic extract of CD
was also analyzed at the same excitation and emission
wavelengths. The scopoletin and mangiferin content
calculated from the standard curve was found to be 4
mg/g and 7 mg/g, respectively.
Thus, a simple analytical method was developed
for determining concentrations of scopoletin and
mangiferin simultaneously in CD. The developed
method was validated for linearity, reproducibility and
accuracy. The linearity was found to be in the range
of 5-30 µg/ml. The correlation coefficients (r) were
0.9919 and 0.9937 for scopoletin and mangiferin,
respectively, indicating good linearity between the
fluorescence intensity and concentration. Scanning
of the samples three times allowed the precision of
the method to be checked. The reproducibility and
accuracy of the method was checked by carrying
out recovery studies. A known concentration of
scopoletin and mangiferin was added to different
concentrations of the methanolic extract i.e. 0.1, 0.2,
0.5 and 1.0 µg/ml. A sample of known concentration
was added in equal volume to the various dilutions
of the extract and analyzed spectrofluorimetrically
to see whether the observed concentration obtained
corresponded to the theoretical concentration
obtained from the standard curve. The percentage
 Table 1. Validation of the spectrofluorimetric method * (percentage recovery of scopoletin and mangiferin)

Concentr- Analyte * content in extract Analyte added Total amount of analyte Amount obtained Percentage recovery
ation of (mg/ml) (mg/ml) expected (mg/ml) (mg/ml) (%)
S.
methanolic
No.
extract used scopoletin mangiferin scopoletin mangiferin scopoletin mangiferin scopoletin mangiferin scopoletin mangiferin
(µg/ml)

1 0.1 0.090±0.031 0.110±0.015 0.097 0.094 0.187±0.031 0.205±0.015 0.189±0.009 0.204±0.045 100.69±1.237 99.51±0.330

228
2 0.2 0.130±0.028 0.149±0.025 0.097 0.094 0.227±0.028 0.243±0.025 0.226±0.009 0.247±0.039 100.69±1.237 101.64±0.238
Medicines, 2008, 3 ( 6 )

3 0.5 0.186±0.011 0.201±0.005 0.097 0.094 0.283±0.011 0.295±0.005 0.288±0.031 0.292±0.039 101.60±0.793 98.98±0.096

4 1.0 0.206±0.011 0.233±0.009 0.097 0.094 0.303±0.011 0.327±0.009 0.304±0.001 0.328±0.004 100.58±0.335 100.30±0.639

* As calculated from the standard curve. All values are Mean ± SD (n = 3).
* Excitation (λmax: 430 nm and 248 nm for scopoletin and mangiferin respectively) and Emission (λ max: 460 nm and 520 nm for scopoletin and mangiferin respectively)
Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional
 Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of Traditional
Medicines, 2008, 3 ( 6 )
recovery of scopoletin and mangiferin was found
to be in the range of (98-102) % (Table 1). Hence,
the developed spectrofluorimetric procedure is a
quick and reliable method for the simultaneous
quantitative monitoring of scopoletin and mangiferin
in raw material, processed powder and in herbal
preparations containing CD.
Acknowledgements
The authors are grateful to Laila Impex Research
Centre, Vijaywada (A.P.), India for generously
providing a gift of scopoletin and would also like to
thank Natural remedies Pvt. Ltd., Bangalore, India for
providing a gift of mangiferin. Two of the authors,
Neeraj Kumar Sethiya and Alok Nahata, would like to
thank the University Grants Commission, New Delhi
for providing junior research fellowships.
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