Industrial Crops and Products 64 (2015) 201208

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Industrial Crops and Products

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An updated method for isolation, purication and characterization of

clinically important antioxidant lignans Sesamin and sesamolin,
from sesame oil
Aejaz Ahmad Dar, Nitish Kumar Verma, Neelakantan Arumugam
Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry 605 014, India

a r t i c l e i n f o a b s t r a c t

Article history: Sesamin and sesamolin are clinically important antioxidant lignans that exhibit anticholestrolemic, anti-
Received 14 June 2014 hypertensive and anticancer properties. Chemically they are phenyl propane dimers synthesized as
Received in revised form 8 September 2014 products of secondary metabolism in several plants. The ancient oil crop Sesamum indicum, by far, is
Accepted 12 October 2014
the major source of these lignans. Several attempts made earlier for isolation of the lignans under con-
Available online 15 November 2014
sideration, in pure form, is far from satisfactory resulting in high cost and their rarity in the market. Here
we report our results on successful isolation and characterization of the two lignans from commercial
Keywords:
sesame oil. A 1:8 mixture of the oil with acetone, on freezing at 80 C, yielded triglycerides and yellow
Sesamin
Sesamolin
oil. The latter when subjected to a similar treatment but with isooctane at 4 C yielded colourless crys-
HPLC talline product. TLC followed by HPLC revealed that the crystalline product is a mixture of 88% sesamin
LCMS and 12% sesamolin. The isolated lignan mixture was subjected to semi-preparative HPLC and TLC to result
NMR in successful separation of putative sesamin and sesamolin in two independent fractions. Purity of the
Lignan diversity compounds thus isolated was conrmed by TLC and LCMS. Structure detail of the isolates was conrmed
by NMR. The sesamolin puried in this study was further tested successfully to prove that it can serve
as reliable biochemical standard for analysis of lignan diversity among sesame germplasm. Relevance of
the method developed for analytical studies and for industrial production of the lignans for therapeutic
purpose is discussed.
2014 Elsevier B.V. All rights reserved.

1. Introduction lignans. By virtue of the antioxidant property these lignans resist

The lignans are products of secondary metabolism produced
by several plants to serve as molecules of defense against the
predators. Chemically they are phenyl propane dimers that addi-
tionally exhibit varied biological activities. Lignans derived from
the ancient oil crop of sesame (Botanical name Sesamum indicum
L.), grown mainly in India, China and Myanmar, by far, is the major
source of clinically important what are known as sesame lignans.
There are eleven different fat soluble aglycon lignans and six dif-
ferent fat insoluble lignan glycosides reported, respectively, from
the sesame oil and oil-free sesame seed meal (Kamal-Eldin et al.,
1994; Moazzami et al., 2006; Kamal-Eldin et al., 2011). Two of the
aglycons namely, sesamin and sesamolin are the major antioxidant
Corresponding author. Tel.: +91 413 2654 544/9944338491;
fax: +91 413 2656 742.
E-mail address: arumugan.dbt@pondiuni.edu.in (N. Arumugam).
rancidity and thereby confer a long shelf life to the sesame oil.
Clinically sesamin exhibit anti-cholesterolemic and antihyper-
tensive properties (Kang et al., 1999; Nakai et al., 2003; Penalvo
et al., 2006; Nishant et al., 2008), and recently it has been asso-
ciated with stimulation of osteoblast differentiation in adipose
stem cells (Wanachewin et al., 2012). Sesamolin on the other hand
showed anticancer property against human lymphoid leukaemia
cells (Miyahara et al., 2001), whereas the minor lignans of sesame
such as sesamol, sesaminol and pinoresinol, inhibited membrane
lipid peroxidation and DNA damage (Osawa, 1999; Kang et al.,
1998; Parihar et al., 2006). Availability of sufcient amount of
individual lignans in pure form at affordable cost is essential for
their application in therapeutics and biochemical analysis. While
pure sesamin standard is expensive, the availability of standard
sesamolin is very rare (Amarowicz et al., 2001; Hemalatha and
Ghafoorunissa, 2004). A review of current literature on sesame
lignans reveal that the focus of current research is primarily on
preparative enrichment of individual lignans in pure form (Sok
et al., 2009), followed by research on mapping the lignan diversity

http://dx.doi.org/10.1016/j.indcrop.2014.10.026
0926-6690/ 2014 Elsevier B.V. All rights reserved.
202 A.A. Dar et al. / Industrial Crops and Products 64 (2015) 201208
in the sesame germplasm for genetic improvement of the crop for
lignan content (Laurentin et al., 2008).
Several attempts have been made earlier for isolation and puri-
cation of individual lignans from sesame oil and seeds (Tracy,
1958; Amarowicz et al., 2001; Hemalatha and Ghafoorunissa, 2004;
Reshma et al., 2010; Zhou et al., 2010). Methods adopted so
far mostly have yielded only a mixture of lignans (sesamin and
sesamolin) rather than their individual harvest in pure form with
the latter continue to be far from satisfactory (Reshma et al., 2010;
Zhou et al., 2010; Chami et al., 2013). Therefore it is imperative
that a protocol be developed for isolation of pure forms of the lig-
nans in industrial scale and their purity authenticated prior to their
employment in medical applications, in studies involving target
based drug development and in analytical biochemistry.
The crop of sesame is represented, globally, by a number of
diverse forms and breeding to enrich the sesamin content in the
crop is one of the prime objectives for sesame breeders (Bhat et al.,
1999; Yasumoto and Katsuta, 2006). Recently we have reviewed
the current status of research on sesame lignans with reference
to their purication methods, biological activities and biosynthesis
(Dar and Arumugam, 2013). Now we present our data on isola-
tion, purication of the two major lignans from sesame oil, their
structural elucidation by nuclear magnetic resonance (NMR) and
utilization of sesamolin isolated as standard in the analysis of
lignans diversity in S. indicum germplasm. To the best of our knowl-
edge this is the rst report to present the structural determination
of the two lignans isolated from sesame oil by NMR and utilization
of pure sesamolin isolated in house in the analysis of sesame lignan
diversity.
2. Materials and methods
2.1. Materials
Idhayam brand commercial sesame oil from the local market of
Pondicherry, India was used as source for isolation of the sesamin
and sesamolin. Sesamin (Cat No. S9314), sesamol (Cat No. S8518)
and naringenin (Cat No. N5893) standards from Sigma Aldrich, AR
grade acetone and isooctane, HPLC grade methanol and water, pre-
coated Kieselgel Silica Gel 60 F254 plates (Cat No. 1.05554.0007)
from Merck and absolute ethanol from Bengal Chemicals, India
were used in the experiments. Sesame varieties with different seed
coat colours, namely, white (Rajeswari, Shekhar, Phuletil, Praghti),
black (Paiyur, Krishna, JLT1, Prachi) and brown (Uma, Nirmala,
Vinayak, Kallika) were procured from National Bureau of Plant
Genetic Resources, New Delhi, and maintained in our Departments
experimental garden were used for lignan diversity analysis.
2.2. Methods
2.2.1. Isolation of lignans from sesame oil
Sesame lignans were isolated from the oil following the method
described in Hemalatha and Ghafoorunissa (2004) with some mod-
ication. Five hundred millilitre of sesame oil was dissolved in
acetone in the ratio of 1:8 (v/v) and incubated for overnight at
80 C. These treatments led to precipitation of triglycerides which
were then separated by vacuum ltration through a 0.45 m nylon
membrane at 4 C. The ltrate was subjected to rotary evaporation
to yield yellow oil. The latter was mixed with isooctane at 1:8 (v/v)
and incubated for 45 days at 4 C to induce crystallization of lig-
nans. The crystalline lignans so obtained were collected by ltration
through Whatman No. 1 lter paper and dried at room tempera-
ture. A pinch of crystals were mounted under a SMZ 1000 Nikon
stereo zoom photomicroscope for observation and representative
crystals were photographed.
2.2.2. Analytical HPLC
High performance liquid chromatography (HPLC) was per-
formed to test the purity of the isolated lignans. A Shimadzu made
HPLC (model LCATVP) equipped with a reverse phase Spincotech
enabled C18 reverse phase column (250 mm 4.6 mm i.d., 5 m
particle size) tted with UV detector (model SPD-10AVP) was used.
The mobile phase consisted of methanol:water (70:30) run at a ow
rate of 0.7 ml/min. The peaks were detected at 290 nm. For HPLC
analysis, the lignan crystal was dissolved in HPLC grade methanol
to make a stock of 1 mg/ml. The lignan solution was ltered through
0.45 m nylon membrane prior to injecting into the HPLC. 10 l
samples of the 0.0001 M of standard sesamin/sesamol/naringenin,
independently as well as in their mixtures were injected into HPLC
to determine their retention time (s).
2.2.3. Semi-preparative HPLC separation of sesamin and
sesamolin
Semi-preparative HPLC was performed to separate sesamin and
sesamolin from the primary lignan crystals into two separate frac-
tions. The same machine described for analytical HPLC above but
tted with a semi preparative reverse phase Phenominex Luna C18
column of dimension 250 mm 10 mm i.d., 10 m particle size
was used. The mobile phase described for analytical HPLC above
was run with a ow rate of 3.1 ml/min (Amarowicz et al., 2001).
300 l of 1 mg/ml solution of the lignan prepared in HPLC grade
methanol was injected into the HPLC. The peaks were detected at
290 nm. As soon as the sesamin peak appeared at 24.34 min on the
monitor of the computer, the fractions were collected manually in
separate reagent bottles repeatedly with 50 injections. Sesamolin
fractions were similarly collected with the appearance of the peak
at 31.75 min. Solvent from the collected fractions was evaporated
using rotary evaporator to obtain powdered fractions of sesamin
and sesamolin in two separate fractions (Fraction I and Fraction II).
2.2.4. Thin layer chromatography (TLC)
TLC was also performed to nd the purity of the isolated com-
pounds. The lignans was dissolved in chloroform at 1 mg/ml and
spotted on TLC plates along with sesamin and sesamol standards.
The TLC was developed with the solvent mixture consisting of
Chloroform, Benzene and Methanol in the ratio 60:40:1 and spots
were visualized in an iodine chamber (Kamal-Eldin et al., 1994).
Alternately the TLCs were visualized under HPTLC-UV analyser. For
obtaining a better yield of sesamin and sesamolin three to four
TLCs were run by loading 1 ml of 10 mg/ml stock sample as a sin-
gle straight streak/band on a 20 cm 20 cm plate and TLC was run
along with commercial sesamin as standard. TLC after development
was visualized under UV and the fraction corresponding to putative
sesamin and putative sesamolin were collected into two separate
vials by scrapping. The lignans were then stripped off the silica by
dissolving in 20 ml chloroform as two separate fractions. The lig-
nans from the two fractions were re-crystallized by evaporation of
the solvent at room temperature and were named putative sesamin
and putative sesamolin.
2.2.5. Liquid chromatographymass spectroscopy (LCMS)
The putative sesamin and sesamolin isolated by TLC were
dissolved in methanol and 10 l of the sample was injected
into Prominence UFLC system (Shimadzu Corporation, Kyoto,
Japan) through a rheodyne sample loop. The system was tted
with a reverse phase C18 Shimpack ODS column (10 4 mm i.d.,
5 m particle size). The sample was eluted isocratically with 70%
methanol run at a constant ow rate of 0.7 ml/min in an auto sam-
pler mode. The eluent was split and introduced into the interface
(ESI probe) of single quadruple mass spectrometer (2.10EV, Shi-
madzu Corporation, Kyoto, Japan). Nitrogen at a ow of 1.5 l/min
was used as nebulizer gas to form a ne spray; big droplets were
 A.A. Dar et al. / Industrial Crops and Products 64 (2015) 201208 203

Fig. 1. Photomicrograph of lignan crystals formed in the isooctane environment as viewed under the microscope (Crystal pool = 10X, Single crystal = 40X).

further regulated by the use of 10 l/min ow of nitrogen drying gas. 3. Results

The analysis was carried out in SCAN mode. The spectral results
were processed with LCMS solution software V 3.4. 3.1. Isolation of lignan crystals from sesame oil and their purity
assessment by HPLC

2.2.6. Nuclear magnetic resonance (NMR) studies
For determination of empirical structure, putative sesamin
and putative sesamolin were dissolved in CDCl3 and subjected
to Fourier transform-nuclear magnetic resonance (Bruker Model:
Avance-Ii) spectroscopy. The NMR was operated with cryomagnet
of eld strength 9.4 T and radio waves of 400 MHz and 100 MHz
applied respectively for obtaining 1 H and 13 C NMR data.
2.2.7. Extraction of lignans from sesame seeds
Lignans from the sesame seeds were extracted following the
protocol of Wang et al. (2012). 100 mg of sesame seeds of each of
the 12 selected varieties were homogenized in pestle and mortar
along with 5 ml of 80% ethanol. The extract was transferred into
30 ml Oakridge tubes and centrifuged for 10 min at 8000 rpm in
Sigma 6K15 centrifuges, set at room temperature. The supernatant
was transferred to a fresh Oakridge tube and the extraction was
repeated. Ethanol was evaporated using rotary evaporator. To the
dry residue, 1 ml of 80% methanol was added and ltered through
0.45 m nylon membrane before HPLC analysis.
The method described in this study yielded on an average of
2.8 g/l of lignans from sesame oil. The yellow oil on treatment with
isooctane at 4 C lead to gradual crystallization of lignans and the
process took 5 days for completing this rst step. The gross view of
the transparent to opaque lignan crystal formed in isooctane envi-
ronment is presented in Fig. 1. The analytical HPLC of the lignan
isolated gave two peaks (Fig. 2). The large peak showed a reten-
tion time (RT) at 23.63 corresponding to the identity of sesamin
standard. The second smaller peak had a RT of 31.71 (Fig. 2). From
the published reports it was inferred that this peak identies with
the RT of sesamolin. Computation of ratio of peak area under the
putative metabolite to the total area of the all the peaks (only two
in our case) in the chromatogram revealed that the lignan crystal
obtained is a mixture consisting of 88% sesamin and 12% sesamolin.
3.2. Preparative separation of sesamin and sesamolin
Semi-preparative HPLC of the isolated lignan was done to sep-
arate sesamin from sesamolin. The process yielded two fractions

2.2.8. Calculation of response factor and determination of

concentration in the unknown sample
0.0001 M each of naringenin as internal standard, sesamin,
sesamol and sesamolin (isolated) as standards were prepared. 10 l
of standards mentioned above were injected, individually as well as
in mixtures, into analytical HPLC to determine the retention time of
the standards and to calculate the response factor following Kupiec
(2004). The response factor (F) was calculated using the formula
F = Ax [S]/As [X] where Ax and As are peak area of reference standard
and internal standard respectively, [X] and [S] are concentration of
reference standard and internal standard respectively in the mix-
ture. For analysis of lignans in the ethanol extract of seeds, the
samples were prepared by mixing of 10 l of the extract with 5 l
of 0.0001 M naringenin. 10 l of this mixture was injected into the
HPLC column. The concentration of sesamin and sesamolin in the
Fig. 2. HPLC of lignan described in Fig. 1 showing presence of two peaks with RT
unknown sample was determined by substitution of values in the of 23.63 min and 31.71 min that correspond to putative sesamin and sesamolin,
formula mentioned above. respectively.
204 A.A. Dar et al. / Industrial Crops and Products 64 (2015) 201208
Fig. 3. An image of TLC of the lignans viewed under 254 nm UV light. The spots
1-5 correspond to sesamol standard, sesamin standard, lignan crystal (Fig. 1),
semipreparative HPLC puried fraction I (sesamin) and fraction II (sesamolin),
respectively.
which were labelled as F-I and F-II. Analytical HPLC of the F-
I showed that it is composed of almost 98.7% pure sesamin as
depicted by a largest peak with a RT of 24.34 and that of the F-II of
highly pure putative sesamolin denoted similarly by a main peak
with 97.4% purity at a RT of 31.75. These results conrmed the suc-
cessful isolation of the sesamin and sesamolin each with more than
97% purity. Similarly TLC of a solution of the isolated lignan crystal
showed presence of two bands indicating it is a mixture and that of
fractions F-I and F-II, from semi-preparative HPLC showed promi-
nent bands corresponding to sesamin and sesamolin respectively
conrming their isolation with utmost purity (Fig. 3). Semi prepar-
ative HPLC though resulted in a reasonably good quality of pure
lignans, the yield turned out to be very low. In order to obtain a bet-
ter yield of putative sesamin and sesamolin an attempt was made to
run a large scale TLC on a 20 cm 20 cm TLC plates in a preparative
scale. Three such runs, on recovery, yielded approximately 20 mg
and 6 mg of putative sesamin and sesamolin respectively. The lig-
nan fractions isolated in such a manner also showed solitary peaks
in HPLC conrming their almost 99% purity (the data not shown).
3.3. LCMS analysis of the putative sesamin and sesamolin
Mass analysis was done to conrm the identity of the com-
pounds isolated by TLC. LCMS of the fraction corresponding
to putative sesamin showed the presence of the characteristic
peak with a m/z of 354 corresponding to m/z ratio of the pure
sesamin (Fig. 4A). Similarly LCMS of the putative sesamolin frac-
tion showed presence of the characteristic peak with m/z of 370
corresponding to m/z of pure sesamolin described in literature
(Fig. 4B). A list of molecular ions deduced form LCMS data is pre-
sented in Table 1. The result indicated that the other compound
present in the primary lignan isolate was sesamolin indeed.
3.4. NMR of the putative sesamin and sesamolin
The 1 H and 13 C NMR of the putative sesamin and putative
sesamolin are presented in Fig. 5AD. Sesamin being a symmet-
ric molecule the 13 C NMR showed 10 peaks representing chemical
shifts of half the number of carbon atoms present in the molecule
(Fig. 5A). Sesamolin is an asymmetrical molecule, and there-
fore showed chemical shifts for all the 20 carbon atoms present
(Fig. 5C). Similarly 1 H NMR showed the presence of 9 and 18 peaks
respectively for sesamin and sesamolin which corresponded to the
respective number of hydrogen atoms present in these molecules
(Fig. 5B and D). The purity and the structural authenticity of the
compounds isolated are thus conrmed.
3.5. Validation of the putative sesamolin as standard in the study
of lignan diversity in sesame germplasm
In order to validate the utility of putative sesamolin isolated
in this study, metabolic diversity of sesame for major lignans was
studied by HPLC analysis of the ethanol extract of the seed. The
major steps involving calibration of HPLC for the standard lignans,
calculation of their response factor using an internal standard, iso-
lation of lignans from the samples and HPLC analysis of the samples
to determine the lignan content are described here.
3.6. HPLC of standards and calculation of response factor
HPLC of internal standard naringenin when run separately gave
single peak with retention time (RT) of 7.11 min. The sesamol,
sesamin and the putative sesamolin isolated in this study gave
peaks with RTs of 6.08, 23.24 and 31.63 min respectively. Similarly
HPLC of the mixture of the standards gave peaks with RTs corre-
sponding to the standards run individually (Fig. 6). The calculated
response factors for the sesamol, sesamin and putative sesamolin
with respect to the internal standard (naringenin) were 0.08, 0.11
and 0.09 respectively. The response factors were used for quanti-
cation of these compounds in the crude lignans isolated from the
seeds of the sesame germplasm.
3.7. HPLC analysis of lignans from seeds of different varieties of S.
indicum L.
The HPLC of the ethanol extracts of sesame seeds with narin-
genin as internal standard depending on the variety showed the
presence of 712 distinct peaks corresponding to as many different
lignans in these samples (Fig. 7). Three of the peaks corresponded to
sesamol, sesamin and sesamolin as revealed by their retention time
coinciding with the respective reference standards (Figs. 6 and 7).
Concentrations of the three compounds were calculated using the
response factor and data is presented in Table 2. A signicant vari-
ability was observed in the content of lignans under consideration
in all the three groups.
4. Discussion and conclusion
Whereas the standard sesamin is commonly available though
with a high cost, commercial sesamolin is very rare. High harvest
of pure lignans is a must for therapeutic purpose and for use as
standard in analytical experiments involving plants and human
subjects (Dar and Arumugam, 2013). In the light of their importance
in human healthcare and economy an investigation on the isolation
of pure forms of these compounds was undertaken. The efforts we
have made to this effect resulted in successful isolation of sesamin
and sesamolin from sesame oil in two independent fractions with
almost 98% purity.
 A.A. Dar et al. / Industrial Crops and Products 64 (2015) 201208 205

Fig. 4. LCMS of fractions I and II described in Fig. 3, showing m/z value of 354 in (A) and 370 in (B) corresponding to sesamin and sesamolin, respectively.

Table 1
List of mass ion fragments deduced from LCMS data of sesamin and sesamolin.

Sesamin m/z Intensity (%) Sesamolin m/z Intensity (%)

M+ 354 4 M+ 370 8
[M+ H]+ 353 12 [M+ H]+ 369 15
[MH H2 O]+ or [M OH]+ 337 100 [M+ CH2 O]+ 340 28
[M+ Ar ]+ 233 8 [M+ Ar ]+ 249 3
[M+ ArCHO+ H ]+ 203 2 [M+ ArOCHO+ H ]+ 203 10
[Ar CH CH CH2 ]+ 161 2 [M+ ArOCHO+ H3 O ]+ 185 100
[ArCO]+ 149 3 [ArOCH2 ]+ 151 2
[ArCH2 + ] 135 2 [ArCO]+ 149 2
Ar+ 121 2 [ArCH2 + ] 135 3
Ar+ 121 2

Where M+ , molecular radical ion; Intensity (%), percentage intensity of m/z signal; Ar, [3,4(methylenedioxy) phenyl] group (C7 H5 O2 ); ArO, [3,4(methylenedioxy) phenoxy]
group.

Table 2
Data on content of prime lignans of seeds of selected varieties of Sesamum indicum deduced from HPLC data.

Seed coat phenotype Name of the sesame variety Lignan content (mg/100 g)

Sesamol Sesamin Sesamolin

Rajeswari 11.64 325.31 161.83

Shekhar 147.95 259.41 113.69
White Phuletil 10.69 384.35 167.48
Praghti 143.44 150.43 76.24
Mean SD 78.43 77.69 279.88 100 129.81 43.10

Uma 4.66 180.74 66.98

Nirmala 51.57 123.31 61.79
Brown Vinayak 15.22 271.40 220.19
Kallika 6.22 244.24 121.21
Mean SD 19.42 21.93 204.92 66.36 117.54 73.52

Paiyur 4.68 307.93 187.61

Krishna 43.07 447.51 328.67
Black JLT1 52.20 184.15 156.21
Prachi 36.87 305.62 314.34
Mean SD 34.20 20.67 311.30 107.65 246.70 87.51
206 A.A. Dar et al. / Industrial Crops and Products 64 (2015) 201208

. Fig. 5A-D The 1 H (B, D) and 13 C (A, C) NMR spectrographs of putative sesamin (A, B) and putative sesamolin (C, D).

Attempts to isolate sesame lignans especially sesamolin have
been made ever since 1940 when the synergistic effect of sesame
oil for the insecticidal pyrethrins was reported (Tracy, 1958). Since
then the main focus has been made to isolate two of the major agly-
con lignans namely, sesamin and sesamolin from varied sources
(Doskotch and El-Feraly, 1969; Baures et al., 1992; Kamal-Eldin
et al., 1994; Amarowicz et al., 2001; Hemalatha and Ghafoorunissa,
2004; Reshma et al., 2010; Zhou et al., 2010). Recently, Williamson
et al. (2008) described some new methods involving photodiode
array detection for sesamin analysis. The isolation of the lignans
involves two major steps. The rst step is either to remove triglyc-
erides, using solvents such as -butyrolactone/n-hexane/acetone

Fig. 6. HPLC of a mixture of standards showing four distinct peaks corresponding to sesamol, naringenin (internal standard), sesamin, and sesamolin (puried) with retention
times of 6.08, 7.39, 23.22 and 31.13 min, respectively.
 A.A. Dar et al. / Industrial Crops and Products 64 (2015) 201208
Fig. 7. HPLC prole of crude ethanol extract of Sesamum indicumvar. Nirmala with the sample run along with the internal standard naringenin showing at least 8 distinct
peaks four of which corresponded to sesamol (RT = 6.50 min), naringenin (RT = 7.51 min), sesamin (RT = 23.70 min) and sesamolin (RT = 32.02 min).
or preferentially dissolve the lignans in aliphatic ethanol or
methanol. The second step involves crystallization of lignans from
the residue obtained from the rst step for which the choices
of solvents used were ethyl acetate/diethyl ether/isooctane. In
our study, we found acetone and isooctane combination to be
a satisfactory one to achieve isolation of good quality lignans.
Though, we have not tested unsaponiable matter (USM) for
lignan content, it is often considered that the USM is enriched
with lignans. Reshma et al. (2010), however, has observed that
the crystallization of lignans directly from methanol extract of
oil yielding 10 g whereas the USM of crystal removed methanol
extract yielding 1 g of lignans per 100 g of oil.
Our observations showed that the crystal resulting in the isooc-
tane environment were good enough to obtain reasonable amount
of sesamin and sesamolin in two separate fractions with qualities
equivalent to that of analytical standards. The quality of primary
lignan crystals that we obtained was at variance with the observa-
tion of Hemalatha and Ghafoorunissa (2004). Their report indicates
these sesamin crystals to be 100% pure but we found it to be a mix-
ture of sesamin and sesamolin as has been reported in many earlier
studies (Chami et al., 2013).
Pure preparation of individual lignan is required for therapeu-
tics, analytical studies and for their structural elucidation. Our
initial attempt to separate sesamin and sesamolin by semi prepar-
ative HPLC (spHPLC) involved repeated injections of a solution of
the impure lignan crystals obtained. Similar activity was reported
by Amarowicz et al. (2001) who adopted spHPLC for separation of
the two compounds from USM. Consumption of high volumes of
solvent and sophisticated instrumentation involved makes lignan
purication by spHPLC an expensive process. Due to low yield of
sesamolin achieved by spHPLC, we relied on TLC for a better yield of
the compound. TLC in fact is simple, fast and does not require much
instrumentation and therefore it could be a method of choice for in
house purication of lignans in small scales.
In most of the cases, the identity of the isolated lignans was
conrmed by mass balance equation rather than NMR. One of
the uniqueness of our report is the structural conrmation of the
sesamin and sesamolin isolated in this study by NMR which con-
rmed further that the isolated compounds were highly pure.
Minor impurities that were present in the preparations of individ-
ual lignans did not interfered with our NMR data and the result fell
in line with the NMR reports of the sesamin from Stachys mialh-
esi (Laggoune et al., 2011) and sesamolin isolated from S. indicum
(Kang et al., 1995).
207
Another signicant contribution of this study is the use of the
putative sesamolin isolated in house, as a standard for estimat-
ing the lignan diversity in the sesame germplasm. S. indicum is the
only major source of the antioxidant lignans under consideration.
Most of the varieties of the crop are poor yielding and require a
lot of agronomic inputs for better yields. Of late efforts are being
made for genetic improvement of the crop through selective breed-
ing (Amarowicz et al., 2001; Zhang et al., 2013). In this regard a
study on lignan diversity in sesame germplasm would highly help
breeders to identify varieties for high lignan content for sesame
breeding. Recently, Lee et al. (2013) described a reasonable method
for quantitative estimation of sesamin and sesamolin in commer-
cial samples by using naphthalene as internal standard instead of
naringenin. Use of this method along with the sesamolin isolated
in our study might result in precise analysis of sesame lignans in
the germplasm as well as in other products.
Preparation of pure sesamin and/or pure sesamolin at industrial
scale is yet to be achieved (Chami et al., 2013). We were able to
isolate nearly 99% pure sesamin and 98% pure sesamolin with good
yield which can be used directly for therapeutic purpose or for use
as standards in lignan analytical studies. For industrial production
of pure sesamin and sesamolin, the primary lignan crystals may
be subjected to column chromatography as reported in Lee and
Choe (2006). We believe that ne tuning of the method reported
herein would surely pave the way for producing the sesamin and
sesamolin at industrial scale which would ultimately result in cost
reduction and their better availability.
Acknowledgements
We acknowledge CIF, Pondicherry University (PU) for NMR facil-
ity, Dr. H. Surya Prakash Rao and his research student Mr. G.
Lakshminarayana of Department of Chemistry, PU for helping in
NMR studies. We also acknowledge UGC-SAP and DBT-PU IPLS for
nancial support.
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