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Industrial Crops and Products 64 (2015) 201208

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Industrial Crops and Products


journal homepage: www.elsevier.com/locate/indcrop

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


rancidity and thereby confer a long shelf life to the sesame oil.
The lignans are products of secondary metabolism produced Clinically sesamin exhibit anti-cholesterolemic and antihyper-
by several plants to serve as molecules of defense against the tensive properties (Kang et al., 1999; Nakai et al., 2003; Penalvo
predators. Chemically they are phenyl propane dimers that addi- et al., 2006; Nishant et al., 2008), and recently it has been asso-
tionally exhibit varied biological activities. Lignans derived from ciated with stimulation of osteoblast differentiation in adipose
the ancient oil crop of sesame (Botanical name Sesamum indicum stem cells (Wanachewin et al., 2012). Sesamolin on the other hand
L.), grown mainly in India, China and Myanmar, by far, is the major showed anticancer property against human lymphoid leukaemia
source of clinically important what are known as sesame lignans. cells (Miyahara et al., 2001), whereas the minor lignans of sesame
There are eleven different fat soluble aglycon lignans and six dif- such as sesamol, sesaminol and pinoresinol, inhibited membrane
ferent fat insoluble lignan glycosides reported, respectively, from lipid peroxidation and DNA damage (Osawa, 1999; Kang et al.,
the sesame oil and oil-free sesame seed meal (Kamal-Eldin et al., 1998; Parihar et al., 2006). Availability of sufcient amount of
1994; Moazzami et al., 2006; Kamal-Eldin et al., 2011). Two of the individual lignans in pure form at affordable cost is essential for
aglycons namely, sesamin and sesamolin are the major antioxidant 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
Corresponding author. Tel.: +91 413 2654 544/9944338491; lignans reveal that the focus of current research is primarily on
fax: +91 413 2656 742. preparative enrichment of individual lignans in pure form (Sok
E-mail address: arumugan.dbt@pondiuni.edu.in (N. Arumugam). 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 2.2.2. Analytical HPLC
lignan content (Laurentin et al., 2008). High performance liquid chromatography (HPLC) was per-
Several attempts have been made earlier for isolation and puri- formed to test the purity of the isolated lignans. A Shimadzu made
cation of individual lignans from sesame oil and seeds (Tracy, HPLC (model LCATVP) equipped with a reverse phase Spincotech
1958; Amarowicz et al., 2001; Hemalatha and Ghafoorunissa, 2004; enabled C18 reverse phase column (250 mm 4.6 mm i.d., 5 m
Reshma et al., 2010; Zhou et al., 2010). Methods adopted so particle size) tted with UV detector (model SPD-10AVP) was used.
far mostly have yielded only a mixture of lignans (sesamin and The mobile phase consisted of methanol:water (70:30) run at a ow
sesamolin) rather than their individual harvest in pure form with rate of 0.7 ml/min. The peaks were detected at 290 nm. For HPLC
the latter continue to be far from satisfactory (Reshma et al., 2010; analysis, the lignan crystal was dissolved in HPLC grade methanol
Zhou et al., 2010; Chami et al., 2013). Therefore it is imperative to make a stock of 1 mg/ml. The lignan solution was ltered through
that a protocol be developed for isolation of pure forms of the lig- 0.45 m nylon membrane prior to injecting into the HPLC. 10 l
nans in industrial scale and their purity authenticated prior to their samples of the 0.0001 M of standard sesamin/sesamol/naringenin,
employment in medical applications, in studies involving target independently as well as in their mixtures were injected into HPLC
based drug development and in analytical biochemistry. to determine their retention time (s).
The crop of sesame is represented, globally, by a number of
diverse forms and breeding to enrich the sesamin content in the 2.2.3. Semi-preparative HPLC separation of sesamin and
crop is one of the prime objectives for sesame breeders (Bhat et al., sesamolin
1999; Yasumoto and Katsuta, 2006). Recently we have reviewed Semi-preparative HPLC was performed to separate sesamin and
the current status of research on sesame lignans with reference sesamolin from the primary lignan crystals into two separate frac-
to their purication methods, biological activities and biosynthesis tions. The same machine described for analytical HPLC above but
(Dar and Arumugam, 2013). Now we present our data on isola- tted with a semi preparative reverse phase Phenominex Luna C18
tion, purication of the two major lignans from sesame oil, their column of dimension 250 mm 10 mm i.d., 10 m particle size
structural elucidation by nuclear magnetic resonance (NMR) and was used. The mobile phase described for analytical HPLC above
utilization of sesamolin isolated as standard in the analysis of was run with a ow rate of 3.1 ml/min (Amarowicz et al., 2001).
lignans diversity in S. indicum germplasm. To the best of our knowl- 300 l of 1 mg/ml solution of the lignan prepared in HPLC grade
edge this is the rst report to present the structural determination methanol was injected into the HPLC. The peaks were detected at
of the two lignans isolated from sesame oil by NMR and utilization 290 nm. As soon as the sesamin peak appeared at 24.34 min on the
of pure sesamolin isolated in house in the analysis of sesame lignan monitor of the computer, the fractions were collected manually in
diversity. 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
2. Materials and methods
using rotary evaporator to obtain powdered fractions of sesamin
and sesamolin in two separate fractions (Fraction I and Fraction II).
2.1. Materials

2.2.4. Thin layer chromatography (TLC)


Idhayam brand commercial sesame oil from the local market of
TLC was also performed to nd the purity of the isolated com-
Pondicherry, India was used as source for isolation of the sesamin
pounds. The lignans was dissolved in chloroform at 1 mg/ml and
and sesamolin. Sesamin (Cat No. S9314), sesamol (Cat No. S8518)
spotted on TLC plates along with sesamin and sesamol standards.
and naringenin (Cat No. N5893) standards from Sigma Aldrich, AR
The TLC was developed with the solvent mixture consisting of
grade acetone and isooctane, HPLC grade methanol and water, pre-
Chloroform, Benzene and Methanol in the ratio 60:40:1 and spots
coated Kieselgel Silica Gel 60 F254 plates (Cat No. 1.05554.0007)
were visualized in an iodine chamber (Kamal-Eldin et al., 1994).
from Merck and absolute ethanol from Bengal Chemicals, India
Alternately the TLCs were visualized under HPTLC-UV analyser. For
were used in the experiments. Sesame varieties with different seed
obtaining a better yield of sesamin and sesamolin three to four
coat colours, namely, white (Rajeswari, Shekhar, Phuletil, Praghti),
TLCs were run by loading 1 ml of 10 mg/ml stock sample as a sin-
black (Paiyur, Krishna, JLT1, Prachi) and brown (Uma, Nirmala,
gle straight streak/band on a 20 cm 20 cm plate and TLC was run
Vinayak, Kallika) were procured from National Bureau of Plant
along with commercial sesamin as standard. TLC after development
Genetic Resources, New Delhi, and maintained in our Departments
was visualized under UV and the fraction corresponding to putative
experimental garden were used for lignan diversity analysis.
sesamin and putative sesamolin were collected into two separate
vials by scrapping. The lignans were then stripped off the silica by
2.2. Methods dissolving in 20 ml chloroform as two separate fractions. The lig-
nans from the two fractions were re-crystallized by evaporation of
2.2.1. Isolation of lignans from sesame oil the solvent at room temperature and were named putative sesamin
Sesame lignans were isolated from the oil following the method and putative sesamolin.
described in Hemalatha and Ghafoorunissa (2004) with some mod-
ication. Five hundred millilitre of sesame oil was dissolved in 2.2.5. Liquid chromatographymass spectroscopy (LCMS)
acetone in the ratio of 1:8 (v/v) and incubated for overnight at The putative sesamin and sesamolin isolated by TLC were
80 C. These treatments led to precipitation of triglycerides which dissolved in methanol and 10 l of the sample was injected
were then separated by vacuum ltration through a 0.45 m nylon into Prominence UFLC system (Shimadzu Corporation, Kyoto,
membrane at 4 C. The ltrate was subjected to rotary evaporation Japan) through a rheodyne sample loop. The system was tted
to yield yellow oil. The latter was mixed with isooctane at 1:8 (v/v) with a reverse phase C18 Shimpack ODS column (10 4 mm i.d.,
and incubated for 45 days at 4 C to induce crystallization of lig- 5 m particle size). The sample was eluted isocratically with 70%
nans. The crystalline lignans so obtained were collected by ltration methanol run at a constant ow rate of 0.7 ml/min in an auto sam-
through Whatman No. 1 lter paper and dried at room tempera- pler mode. The eluent was split and introduced into the interface
ture. A pinch of crystals were mounted under a SMZ 1000 Nikon (ESI probe) of single quadruple mass spectrometer (2.10EV, Shi-
stereo zoom photomicroscope for observation and representative madzu Corporation, Kyoto, Japan). Nitrogen at a ow of 1.5 l/min
crystals were photographed. 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 The method described in this study yielded on an average of
For determination of empirical structure, putative sesamin 2.8 g/l of lignans from sesame oil. The yellow oil on treatment with
and putative sesamolin were dissolved in CDCl3 and subjected isooctane at 4 C lead to gradual crystallization of lignans and the
to Fourier transform-nuclear magnetic resonance (Bruker Model: process took 5 days for completing this rst step. The gross view of
Avance-Ii) spectroscopy. The NMR was operated with cryomagnet the transparent to opaque lignan crystal formed in isooctane envi-
of eld strength 9.4 T and radio waves of 400 MHz and 100 MHz ronment is presented in Fig. 1. The analytical HPLC of the lignan
applied respectively for obtaining 1 H and 13 C NMR data. 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
2.2.7. Extraction of lignans from sesame seeds the published reports it was inferred that this peak identies with
Lignans from the sesame seeds were extracted following the the RT of sesamolin. Computation of ratio of peak area under the
protocol of Wang et al. (2012). 100 mg of sesame seeds of each of putative metabolite to the total area of the all the peaks (only two
the 12 selected varieties were homogenized in pestle and mortar in our case) in the chromatogram revealed that the lignan crystal
along with 5 ml of 80% ethanol. The extract was transferred into obtained is a mixture consisting of 88% sesamin and 12% sesamolin.
30 ml Oakridge tubes and centrifuged for 10 min at 8000 rpm in
Sigma 6K15 centrifuges, set at room temperature. The supernatant 3.2. Preparative separation of sesamin and sesamolin
was transferred to a fresh Oakridge tube and the extraction was
repeated. Ethanol was evaporated using rotary evaporator. To the Semi-preparative HPLC of the isolated lignan was done to sep-
dry residue, 1 ml of 80% methanol was added and ltered through arate sesamin from sesamolin. The process yielded two fractions
0.45 m nylon membrane before HPLC analysis.

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

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

Fig. 3. An image of TLC of the lignans viewed under 254 nm UV light. The spots HPLC of internal standard naringenin when run separately gave
1-5 correspond to sesamol standard, sesamin standard, lignan crystal (Fig. 1), single peak with retention time (RT) of 7.11 min. The sesamol,
semipreparative HPLC puried fraction I (sesamin) and fraction II (sesamolin), sesamin and the putative sesamolin isolated in this study gave
respectively.
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
which were labelled as F-I and F-II. Analytical HPLC of the F- response factors for the sesamol, sesamin and putative sesamolin
I showed that it is composed of almost 98.7% pure sesamin as with respect to the internal standard (naringenin) were 0.08, 0.11
depicted by a largest peak with a RT of 24.34 and that of the F-II of and 0.09 respectively. The response factors were used for quanti-
highly pure putative sesamolin denoted similarly by a main peak cation of these compounds in the crude lignans isolated from the
with 97.4% purity at a RT of 31.75. These results conrmed the suc- seeds of the sesame germplasm.
cessful isolation of the sesamin and sesamolin each with more than
97% purity. Similarly TLC of a solution of the isolated lignan crystal
3.7. HPLC analysis of lignans from seeds of different varieties of S.
showed presence of two bands indicating it is a mixture and that of
indicum L.
fractions F-I and F-II, from semi-preparative HPLC showed promi-
nent bands corresponding to sesamin and sesamolin respectively
The HPLC of the ethanol extracts of sesame seeds with narin-
conrming their isolation with utmost purity (Fig. 3). Semi prepar-
genin as internal standard depending on the variety showed the
ative HPLC though resulted in a reasonably good quality of pure
presence of 712 distinct peaks corresponding to as many different
lignans, the yield turned out to be very low. In order to obtain a bet-
lignans in these samples (Fig. 7). Three of the peaks corresponded to
ter yield of putative sesamin and sesamolin an attempt was made to
sesamol, sesamin and sesamolin as revealed by their retention time
run a large scale TLC on a 20 cm 20 cm TLC plates in a preparative
coinciding with the respective reference standards (Figs. 6 and 7).
scale. Three such runs, on recovery, yielded approximately 20 mg
Concentrations of the three compounds were calculated using the
and 6 mg of putative sesamin and sesamolin respectively. The lig-
response factor and data is presented in Table 2. A signicant vari-
nan fractions isolated in such a manner also showed solitary peaks
ability was observed in the content of lignans under consideration
in HPLC conrming their almost 99% purity (the data not shown).
in all the three groups.

3.3. LCMS analysis of the putative sesamin and sesamolin 4. Discussion and conclusion

Mass analysis was done to conrm the identity of the com- Whereas the standard sesamin is commonly available though
pounds isolated by TLC. LCMS of the fraction corresponding with a high cost, commercial sesamolin is very rare. High harvest
to putative sesamin showed the presence of the characteristic of pure lignans is a must for therapeutic purpose and for use as
peak with a m/z of 354 corresponding to m/z ratio of the pure standard in analytical experiments involving plants and human
sesamin (Fig. 4A). Similarly LCMS of the putative sesamolin frac- subjects (Dar and Arumugam, 2013). In the light of their importance
tion showed presence of the characteristic peak with m/z of 370 in human healthcare and economy an investigation on the isolation
corresponding to m/z of pure sesamolin described in literature of pure forms of these compounds was undertaken. The efforts we
(Fig. 4B). A list of molecular ions deduced form LCMS data is pre- have made to this effect resulted in successful isolation of sesamin
sented in Table 1. The result indicated that the other compound and sesamolin from sesame oil in two independent fractions with
present in the primary lignan isolate was sesamolin indeed. 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 et al., 1994; Amarowicz et al., 2001; Hemalatha and Ghafoorunissa,
been made ever since 1940 when the synergistic effect of sesame 2004; Reshma et al., 2010; Zhou et al., 2010). Recently, Williamson
oil for the insecticidal pyrethrins was reported (Tracy, 1958). Since et al. (2008) described some new methods involving photodiode
then the main focus has been made to isolate two of the major agly- array detection for sesamin analysis. The isolation of the lignans
con lignans namely, sesamin and sesamolin from varied sources involves two major steps. The rst step is either to remove triglyc-
(Doskotch and El-Feraly, 1969; Baures et al., 1992; Kamal-Eldin 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 207

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