American Journal of Plant Sciences, 2010, 1, 12-16
doi:10.4236/ajps.2010.11002 Published Online September 2010 (http://www.SciRP.org/journal/ajps)
TLC Determination of Marmesin, a Biologically
Active Marker from Feronia Limonia L.
Mahendra Jain*, Ashish Trivedi, S.H.Mishra
Herbal Drug Technology Laboratory, Pharmacy Department, Faculty of Technology and Engineering, The M. S. University of
Baroda, Kalabhavan, Vadodara, Gujarat, India.
Received August 9th, 2010; revised August 24th, 2010; accepted September 6th, 2010
ABSTRACT
Feronia limonia Linn. (Rutaceae) have gained traditiona l therapeutic importance owing to their high essential oil and
coumarins content. Marmesin, a furanocoumarin was identified by TLC and isolated by column chromatography and
further purified by Preparative TLC. Presently, there is no app ropriate TLC based method available for standardization
of F. limonia. A simple, sensitive and accurate high performance thin layer chromatographic (HPTLC) method has
been developed for the estimation of marmesin in the methanolic extract of stem bark of Feronia limonia. HPTLC was
performed on precoated silica gel 60F254 aluminium plates (20 cm × 20 cm) with Chloroform: Methanol (9.5:0.5), as
mobile phase. Quantitative evaluation of the plate was performed in the absorption-reflection mode at 338 nm. The
calibratio n curve was linear in the concentra tion range of 20 – 100 ng spo t–1. The method was validated for precision,
repeatability an d accuracy. The tech nique has been app lied, for the first time, for the estimation of marmesin. The pro-
posed method was found to be robust, precise, and accurate, it therefore holds potential for detection, monitoring and
quantification of ma rmesin in Feronia limonia and its related formulation.
Keywords: Marmesin, Feronia Limonia, HP TLC
1. Introduction
Standardization and characterization of herbal drugs is a
topic of continuous scientific interest in the herbal drug
industry [1]. With the advent of modern chromatographic
systems there is an ever increasing intent to produce and
develop easy, rapid, convenient and cost effective metho-
ds for standardization of herbal drugs based on their phy-
toconstituents. This requirement is fulfilled by thin layer
chromatography (TLC) [2,3]. Feronia limonia is (family
Rutaceae, subfamily Aurantioideae), commonly known
as wood-apple , belongs to the tribe Citreae and subtribe
Balsamocitrinae[4] which is widely distributed in dry wa-
rm regions of India, Bangladesh, Barma, Ceylon, Java &
Srilanka [5,6]. This plant recently gained a great thera-
peutically relevance owing to their high Coumarins and
monoterpenoids content, which is explored for treatment
of snake bite [7]. Stem bark mainly consists of furan Al-
kaloid; Coumarins; Flavanones; Lignan; triterpene [8]. It
is useful as tonic in diarrhoea, dysentery, stomatitis, tum-
ors, cough, asthma, leucorrhoea, wounds and ulcers. Fru-
its, leaves and stem bark of F. limonia have been studied
for anti-tumor [9], larvicidal [10] and antimicrobial ac-
tivity [8].
Marmesin is one of the most prevalent linear dihydro-
furanocoumarin, is abundant in species belonging to the
families of Umbelliferae, Apiaceae, Rutaceae, Moraceae,
and Leguminoseae [11,12]. It is originally isolated from
indigenous indian plants, Aegle marmelos Correa [13],
and later from the Hawiian shrub Pelea barbigera [14]
both of these are from rutaceae family. It has an amazing
array of scientifically acknowledged benefits for key ar-
eas of health, as dermal photosensitizing activity benefi-
cial in the treatment of leucoderma [15], antifungal activ-
ity [16], phytoalexin [17], feeding deterrence effects [18]
and radical scavenging activity [19]. Currently HPTLC is
often used as an alternative to HPLC for the quantifica-
tion of plant products because of its simplicity, accuracy,
cost-effectiveness and rapidity [20]. The present study is
based on development of methods for determination of
marmesin by HPTLC in F. limonia stem bark that may
contribute in standardization of raw material of the plant
and its formulation.

TLC Determination of Marmesin, a Biologically Active Marker from Feronia limonia L.
13
2. Experimental
2.1. Reagents and Chemicals
All the chemicals, including solvents, were of analytical
grade from E. Merck, India. The HPTLC plates Si 60F254
(20 cm × 20 cm) were purchased from E. Merck (Darm-
stadt, Germany).
2.2. Plant Materials
The plant material of Feronia limonia was collected in
the months of September– October 2008 from campus of
The M.S. University, Vadodara (Gujarat). They were
authenticated in the Botany Department and a voucher
specimen (No.Pharmacy/FL/ 08-09/01/MJ) has been de-
posited in the Pharmacy Department of The M. S. Uni-
versity of Baroda, Vadodara, India.
2.3. Extraction and Isolation of Reference
Compound (MR-1) from Feronia Limonia
Air-dried and finely powdered stem barks of the plant
(500 g) were exhaustively extracted at temperature (60-
80˚C) with methanol (3 × 1.5 L) in a soxhlet apparatus
and the pooled extracts then obtained were concentrated
under vacuum to give methanolic extract. Methanolic ex-
tract was made hydroalcoholic by addition of hot dis-
tilled water in 1:1 ratio partitioned with chloroform (100
mL × 4), and combined chloroform fraction was concen-
trated in vacuum to afford a brown residue (4.5 g). This
residue was chromatographed over a Silica gel (60#120
mesh size) column eluting with toluene followed by in-
creasing concentrations of ethyl acetate and methanol.
Fraction 9-10 (toluene: ethyl acetate, 60:40) yielded yel-
lowish crystal resulted in mixture of compounds on TLC.
Further purification of MR-1 was achieved by prepara-
tive TLC (chloroform: methanol, 9.8:0.2) and confirmed
by analytical HPLC. MR-1 obtained as white crystal (118
mg). The structure elucidation of MR-1 was performed
with the help of 13CNMR, mass (ESI-MS) spectra and
CHN analysis that confirmed as marmesin reported earl-
ier [2,3-dihydro-2-(1-hydroxy-1 methylethyl)- 7H-furo[3,
2-g][1]benzopyran-7-one] (Figure 1)[21].
Marmesin (MR-1): C14H14O4 m.p.188-1900 (CHCl3-pe-
trol); IR spectra: 3479,2977, 2929, 1703, 1630, 1572,
14851444, 1404 and 819 cm-1 ;1H NMR: δ1.23 and
1.37(> CMe2, 1.85(1H, br), 3.23 (2H, br d, J 8.8 Hz,
H2-1’), 4.74 (1H, t, J 8.8 Hz, H-2’), 6.21(1H, d, J 9.5 Hz,
H-3), 6.74(1H, s, H-8), 7.22(1H, s, H-5), 7.59(1H, d, J
9.5 Hz, H-4) ; m/z (%) 246 (M+,39), 213(20), 188 (75),
187(100), 175(15), 160(30), 131(19), 59(66), 43(7) ;
CHO % elements- (Oxyzen-25.915), (Carbon-67.191)
and (Hydrozen-5.480).
2.4. Preparation of Crude Extract
Accurately weighed 5 g of the coarse powder of F. limo-
nia stem barks were extracted with methanol (3 × 50 mL)
under reflux (30 min each time) on a water bath. The co-
mbined extracts were filtered and concentrated, and tra-
nsferred to a 25 mL volumetric flask and the volume was
made up with methanol.
2.5. Preparation of Standard Solution
A stock solution of marmesin (100 µg mL-1) was prep-
ared by dissolving 1 mg of accurately weighed marmesin
in methanol and making up the volume of the solution to
10 mL with methanol.
2.6. Chromatography
A Camag TLC system equipped with Camag Linomat V
an automatic TLC sample spotter, Camag glass twin tro-
ugh chamber (20 × 10 cm), Camag scanner 3 and integra-
ted win CATS 4 Software were used for the analysis. TLC
was performed on a pre-coated TLC plate silica gel60F254
Figure 1. Mass spectroscopy and chemical structure of marmesin.

TLC Determination of Marmesin, a Biologically Active Marker from Feronia limonia L.
14
(20 cm × 10 cm). Samples and standards were applied on
the plate as 8 mm wide bands with an automatic TLC
sampler (Linomat V) under a flow of N2 gas, 10 mm
from the bottom and 10 mm from the side and the space
between two spots were 15 mm of the plate. The linear
ascending development was carried out in a Camag twin
trough chamber (20 cm × 10 cm) which was presa- tu-
rated with 20 mL mobile phase chloroform: methanol
(9.5:0.5 v/v) for 20 min at room temperature (25 ± 2˚C
and 40% relative humidity). The length of the chroma-
togram run was 9 cm. Subsequent to the development,
TLC plates were dried under stream of hot air and then
subjected to densitometric scanning using a Camag TLC
scanner III (Camag, Switzerland) with win CATS soft-
ware (version 1.4.1) in the absorbance- reflectance scan
mode. Quantitative evaluation of the plate was performed
in absorption-reflection mode at 338 nm. Quantification
of marmesin in the extract of F. Limonia stem barks was
performed by external standard method, using pure mar-
mesin as standard.
2.7. Calibration Curve for Marmesin
Stock solution of marmesin (100 µg mL-1) was prepared
in methanol and different amounts (20–100 ng spot-1)
were applied on a TLC plate, using Linomat V for pre-
paring five point calibration graphs of peak area versus
concentration. The regression equation for marmesin was
1089.554 + 230.603x and co-relation coefficient (r2) was
0.999.
2.8. Quantification of Marmesin in Test Sample
Ten microlitres of sample solution were applied in tripli-
cate on a TLC plate and developed, scanned as above.
Peak areas were recorded and the amount of marmesin
was calculated using the calibration plot.
2.9. Specificity
The specificity of the method was ascertained by co-an-
alyzing standard and sample. The band for marmesin in
sample was confirmed by comparing the Rf (0.49) and
absorption spectra of the spot to that of reference comp-
ound. The peak purity of marmesin peak in sample track
was assessed by comparing the spectra at peak start, peak
apex and peak end positions of the band. Good correla-
tion was also obtained between standards and sample
overlay spectra (r2 > 0.99).
2.10. Method Validation
The method was validated for precision, accuracy and
repeatability [22]. Instrumental precision was checked by
repeated scanning of the same spot 20 and 100 ng five
times and was expressed as coefficient of variance (%
RSD). Method precision was studied by analyzing the st-
andards 20 and 100 ng per spot under the same analytical
procedure and lab conditions on the same day and on
different days (inter-day precision) and the results were
expressed as % RSD. Accuracy of the method was tested
by performing the recovery studies of the pre-analysed
sample with standard at three levels (55.02, 68.78 and
82.53 µg mL-1), % recovery and average % recovery
were calculated.
3. Result and Discussion
Chloroform: methanol (9.5:0.5 v/v) gave the best resolu-
tion and satisfactory separation of the components in the
extracts with well resolved peaks. A total of nine peaks
were observed methanol extracts of samples. A compara-
tive chromatographic display is shown in Figures 2(a) &
2(b). The densitometric scanning was therefore performed
at a wavelength of 338 nm. The identities of the bands of
marmesin (Rf = 0.49), in the sample extract were con-
firmed by overlaying their absorption spectra with those
of the standard compounds using the TLC Scanner 3.
The peak purity of the separated marmesin was confir-
med by recording the absorption spectra at start to middle
and middle to the end of the peak.
(a) (b)
Figure 2. (a) TLC chromatogram, for a standard marmesin in methanol, Calibration curve and Three-dimensional overlaid
chromatogram of standard track and sample track for marmesin; (b) TLC chromatogram, for stem barks methanolic extract
of Feronia Limonia.

TLC Determination of Marmesin, a Biologically Active Marker from Feronia limonia L.
15
3.1. System Suitability Test
3.1.1. Linearity and Detection Limit
Linearity was checked by applying standard solutions of
marmesin at five different concentration levels. The cali-
bration curve was drawn in the concentration range of
20–100 ng spot-1(Figures 2(a) and 2(b)). The equation
for the calibration curve of marmesin is Y = 1089.554 +
230.603x and the correlation coefficient of the calibration
plot was 0.999 indicating good linearity. Results of re-
gression analysis on the calibration curve and detection
limits are presented in Table 1(a).
3.1.2. Precision Studies
Instrumental precision was checked by repeated scanning
of the same spots (20 and 100 ng spot-1) of standard ma-
rmesin five times and the RSD values were 1.56 and 1.82
for 20 and 100 ng spot-1, respectively. To determine the
precision of the developed assay method 20 and 100 ng
spot-1 of the marmesin standard was analysed five times
within the same day to determine the intra-day variability.
The RSD values were 3.41 and 6.29 for 20 and 100 ng
Table 1. Method validation parameters for quantification of
marmesin using proposed TLC densitometric method.
(a) Linearity regression data
Sl no. Parameter Results
1 RF 0.49
2 Dynamic range (ng spot-1) 20–100
3 Equation Y = 1089.554 + 230.603x
4 Slope 230.603
5 Intercept 1089.554
6 Limit of detection 5 ng
7 Limit of quantification 15.15 ng
8 Linearity (correlation coefficient) 0.999
9 Specificity Specific
(b) Precision studies data
Method precision (% RSD)
Concentration
(ng spot-1)
Instrumental precision
(% RSD) Intra-day Inter-day
20 1.56 3.41 2.68
100 1.82 6.29 2.83
(c) Recovery studies of marmesin
Sl no.
Amount of marmesin
present in the sample
(µg)
Amount
added
(µg)
Amount
found
(µg)
Avg.
Recovery
(%)
1 68.78 55.02 122.11 98.83
2 68.78 68.78 136.92
3 68.78 82.53 148.81
spot-1, respectively. Similarly the inter-day precision was
tested on the same concentration levels on 2 days and the
RSD values were 2.68 and 2.83, respectively (Table 1(b)).
3.1.3. Sampl e Analysis and Recovery Studie s
This developed TLC method was subsequently applied
for the analysis of marmesin in the methanolic extract of
Feronia limonia stem barks. The marmesin content of the
stem barks by this proposed method was found to be
0.03412%. For the examination of recovery rates, 80,
100 and 120% of pure marmesin were added to preana-
lyzed sample and quantitative analysis was performed.
The average recovery was 98.83 (Table 1(c)).
4. Conclusions
Thin layer chromatography is a globally accepted, ratio-
nal and practical solution to characterize the crude plant
drug along with pharmacologically active constituent en-
riched standardized extracts and their formulations. TLC
method on silica gel 60F254 with chloroform–methanol
(9.5:0.5, v/v) was developed and densitometric evalua-
tion was performed at 338 nm. This method is simple,
specific, precise, accurate and robust for the determina-
tion of marmesin [2,3-dihydro-2-(1-hydroxy-1 methylethyl)-
7H-furo[3,2-g][1]benzopyran-7-one]. This standardized
TLC procedure may be used effectively for the screening
analysis as well as quality evaluation of the plant or its
derived herbal products.
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