Identification and Exploration of Lecithin from Novel Sources and its Applications

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INTRODUCTION
Quail is a small size bird that belongs to pheasant family
mainly feed on small insects and plants. There are nearly 45
varieties of Quail species present around the world, among
which Coturnix Japonica1 a brown coloured Quail variety is
mainly bred in Indian farms for their meat and eggs. Quail’s
egg is smaller in size, with thin shells and brown spots over it
having the weight in the range of 100-150 g (Fig. 1). It consists
of 45 % of yellow yolk of its total content which is higher in
proportion compared to hen’s egg. They are considered to serve
as a novel source of lecithin.
Lecithin source
Fig. 1. Quail’s egg
Lecithin is complex mixture of phospholipids (PL), fatty
acids, carbohydrates with major constituent being phospha-
tidylcholine compound. Phospholipids grabs much attention
because of their unique characteristic and simplicity in use.
They are mainly used in pharmaceutical industries for the
preparation of creams, gels and other formulations. They have
Identification and Exploration of Lecithin from Novel Sources and its Applications†
S. BALAGURU, D. RAMYA DEVI and B.N. VEDHA HARI*
Department of Pharmaceutical Technology, School of Chemical and Biotechnology, SASTRA University, Thanjavur-613 401, India
*Corresponding author: Fax: +91 4362 264120; Tel: +91 4362 264101/108 Extn: 116; E-mail: vedhahari@scbt.sastra.edu
Published online: 5 June 2014; AJC-15336
Recent research on natural sources such as plants, microbes and other naturally derived compounds have grabbed much attention because
of their reduced side effects in treating various ailments and biocompatibility. Technological advancements in the field of medicine have
additionally triggered the researchers in developing novel systems for the treatment of many diseases among which cancer research has
gained much importance in recent years. Lecithin, a naturally occurring compound present in egg yolk, soya bean oil and milk possess
significant anticancer and antioxidant activity. It is composed of phospholipids, fatty acids, carbohydrates and a major constituent of
phosphatidylcholine molecule, which is responsible for the normal functioning of nerve cells and cell membranes. The objective of the
present work is to extract lecithin compound from Quail’s egg using chemical extraction and its characterization using thin layer
chromatography, gas chromatography-mass spectroscopy and nuclear magnetic resonance spectroscopy for the structural elucidation of
various constituents present in the crude lecithin. Further, the extracted crude lecithin has been assayed for cytotoxic activity in normal
cell line using MTT assay.
Keywords: Lecithin, Phosphatidylcholine, Cytotoxicity, MTT assay.
†Presented at PHYTOCONGRESS-2013, held on 7-8 March 2013, SASTRA University, Thanjavur, India
wide applications in cosmetic and food industries too. Soya
lecithin one of the major sources of phospholipids is obtained
as by-product from oil refining industries which are highly
impure in nature and so search of alternative sources for lecithin
lead the researchers for the use of eggs. The main aim and
objective of this work is to separate lecithin from Quail egg and
to characterize the extracted crude lecithin qualitatively and
quantitatively using TLC, GC-MS, NMR, FTIR and DSC
analytical techniques and finding their potential uses in various
therapeutic applications.
EXPERIMENTAL
Quail’s eggs were purchased from Aishwarya quail farm,
Pondicherry. Chemicals such as silica gel, acetone, chloroform
and methanol used were of analytical grade.
Lecithin isolation from quail egg yolk: Modified single-
ton gray procedure was followed to isolate lecithin from quail
egg yolk. Initially eggs were collected and needed yellow yolk
was separated manually, followed by chemical extraction
method for the isolation of lecithin2. To the yolk, solvent mixture
of chloroform:methanol in the ratio of 2:1 was added and the
mixture was stirred for 2 h at 200 rpm under room temperature.
Then the mixture was transferred into separating flask and
kept undisturbed for 1 h, later the clear solution settling down
ASIAN JOURNAL OF CHEMISTRY
ASIAN JOURNAL OF CHEMISTRY
Asian Journal of Chemistry; Vol. 26, No. 12 (2014), 3736-3740
http://dx.doi.org/10.14233/ajchem.2014.17065

would contain the required lecithin compound which was
collected separately. This solution was at last concentrated
using ice cold acetone to make the required lecithin to get
precipitated out3,4. Using vaccum filtration techniques preci-
pitated lecithin was filtered and the isolated mass was stored
in amber colour bottle and kept in deep freezer at (-20 ºC)
until further use5,6.
Qualitative analysis of lecithin by TLC: Thin layer chro-
matography technique was performed using laboratory
procedures. Glass slides were thoroughly cleaned with distilled
water followed by ethanol and were dried. Silica gel slurry
was prepared by mixing appropriate amount of silica with
distilled water and made into paste using mortar and pestle.
Prepared slurry was uniformly poured on the glass slides to
get a uniform thickness of the TLC plate. Solvent system of
chloroform:methanol:water of 65:25:4 ratio was prepared and
the TLC chamber was saturated with solvent mixture before
starting the experiment for the detection of lecithin7. Both the
isolated lecithin and soya lecithin (reference standard for crude)
were loaded as small spot on the prepared plate and was kept
in the saturated solvent chamber for run8. The solvent was
allowed to move through till three-fourth of the plate. Then
they were removed, air dried and placed in iodine chamber
for the detection of the sample.
f
Distance travelled by the substance
R value
distance moved up by the solvent
=
Gas chromatography-mass spectroscopy (GC-MS): Gas
chromatography mass spectroscopy was done to detect the
compounds present in the crude lecithin sample. GC-MS
analysis was performed by injecting 1 µL of sample into the
oven, which was dissolved in suitable chloroform:methanol
(2:1) solvent with helium as a carrier gas maintained at constant
flow rate. GC-MS oven was programmed from 70-260 ºC
which increases at the rate of 10 ºC/min. Mass spectroscopic
analysis was done with an electron ionization at a voltage of
70 eV and the whole process was carried out for 40 min for
complete analysis. Finally spectral results of crude sample were
obtained with respect to retention time and peak area.
Nuclear magnetic resonance spectroscopy (NMR): NMR
spectroscopy is based on the phenomena of nuclear magnetic
resonance which gives elaborate information about structural
and chemical properties of various compounds present in the
crude sample. NMR of both carbon (13C) and proton (1H ) were
done in order to find carbon and hydrogen atoms containing
functional groups of compounds and their pattern of arrange-
ment in them which can provide a clear idea of the crude
sample. NMR spectroscopy was performed by dissolving
extracted crude lecithin in chloroform (deuterium as isotope
of hydrogen) solvent for analysis. External magnetic field was
applied under reduced pressure in order to get chemical shift
of the compounds9. Based on the nuclear magnetic resonance
frequencies, molecules in the compound experiences different
spins of hybridization and undergoes different stretches which
gives the complete structural elucidation of the crude sample.
Fourier transform infrared spectroscopy (FT-IR): Fourier
transform infrared spectroscopy was done to find interactions
and compatibility between molecules in the sample. Analysis
was performed using ATR techniques, which was based on
the aspect of measuring the intensity of internally reflected
beams from the samples. The procedure consists of dispersing
the sample into KBr and it was compressed to thin film using
high pressure. Later these films were placed in between the cells
of two IR transparent windows for total internal reflections11,12.
Lecithin sample was made into a thin film and it was placed in
between the cells, it was scanned in the wave number range of
4000-400 cm-1 and the output spectra obtained were recorded.
Differential scanning calorimetry (DSC-TGA): DSC
was performed to find the exothermic and endothermic changes
taking place in sample with respect to rise in temperature. It
gives information like glass transition temperature, recrystalli-
zation and melting point of the sample. TGA was performed
to find the weight loss of the sample with respect to time and
temperature increase. They also help in determining the thermal
stability and degradation nature of the sample as a function of
heat. The process was carried out by placing 3-8 mg of crude
lecithin in alumina pan under nitrogen (N2) atmosphere as
carrier gas with flow rate of 30 mL/min at a scanning rate of
10-500 ºC/min. Thermograms of sample were obtained with
distant peaks with respect to temperature11,13.
Cytotoxicity efficacy of lecithin: MTT assay is a colouri-
metric assay done to determine the viability of cells in vitro
condition14. MTT assay was done for crude lecithin to check
the cytotoxicity level using NIH3T3 normal cell line at various
concentrations. NIH3T3 cell lines were seeded in 96 well plates
and incubated for 24 h. Lecithin was loaded in the plate and
incubated for 1 h, later plates were removed from incubator
and 10 µL of MTT was added and incubated for 4 h. Formazan
crystals were formed in the plate and to that 100 µL of isopropyl
alcohol was added and kept in shaker till it gets dissolved.
The solution was separated and analyzed for absorbance at
570 nm.
100
Control
TestControl
(%) Inhibition ×
−
=
RESULTS AND DISCUSSION
Qualitative analysis: Brownish yellow colour spots in
the TLC plate confirmed the compounds of quail egg. Lecithin
detected is equivalent to soya lecithin which was used as refe-
rence standard. Rf value was calculated for both the detected
egg lecithin and standard soya lecithin and it was found to be
similar in the range of 0.8 (Fig. 2). Isolated compound from
quails egg was proved to be lecithin with reference 2.
Molecular mass determination: The output spectrum
from GC-MS was analyzed and the identified compounds were
given in Table-1. Compounds in the crude sample were identi-
fied based on the comparison of spectral data with that of
standard compounds and by matching with that to NIST library
software preloaded. Spectral peak area gave an idea of probable
compounds being present in the sample. It was seen that deri-
vatives of palmatic acid and Stearic acid were found to be in
the percentage range of 61.67 and 43.3 which were the group
of fatty acids and triglycerides confirming that the isolated
mass contains the compounds of lecithin. Structures of these
compounds are shown in Fig. 3a,b and the GS-MS spectra of
crude lecithin is given in Fig. 4.
Vol. 26, No. 12 (2014) Identification and Exploration of Lecithin from Novel Sources and its Applications 3737

A - Qualis egg lecithin (Test)
B - Soya lecithin (Standard)
Fig. 2. TLC profile of crude lecithin with soya lecithin as standard
TABLE-1
COMPOUNDS DETECTED IN CRUDE
LECITHIN USING GC-MS ANALYSIS
Compound name m.w. m.f. Probability
4,6-Dimethoxy-7-(5-methyl-1-
pyrrolin-2-yl)-2,3-diphenylindole
410 C27H26N2O2
81.68
Hexadecanamide, N-(2-
hydroxyethyl)
299 C18H37NO2 79.56
Isopropyl dodecanoate 242 C15H30O2 79.19
Hexadecanoic acid, methyl ester 270 C17H34O2 61.67
Tetradecane 198 C14H30 55.53
Pentadecane 212 C15H32 54.51
Dotriacontane 450 C32H66 52.52
Ether, methyl 1-octadecenyl 282 C19H38O 43.3
Heneicosane 296 C21H44 35.84
Dodecanamide, N-(2-
hydroxyethyl)
243 C14H29NO2 35.71
Isochiapin 350 C19H26O6 25.50
Octacosane 394 C28H58 21.96
HO
O(a)
O
O
OH
(b)
Fig. 3. (a) Structure of palmitic acid. (b) Structure of stearic acid
Nuclear magnetic resonance spectroscopy (NMR):
Peaks obtained from the carbon 13C and proton 1H NMR (Fig. 5)
revealed the presence of carbon and hydrogen atom containing
compounds in them. From 13C NMR spectra chemical shifts
in the range of 14.24-54.39 ppm confirmed the presence of
non polar alkyl and ester compounds and shift at 76.74-77.59
ppm indicated the presence of carbohydrates groups in them.
In proton 1H NMR spectra the chemical shift from 0.837-7.261
10 15 20 25 30 35 40
Time (min)
100
95
90
85
80
75
70
65
60
55
50
45
.40
35
30
25
20
15
10
5
0
R
e
l
a
t
i
v
e
a
b
u
n
d
a
n
c
e
6.28
10.98
12.73
14.62
18.74
19.19
22.26
23.88
24.71
27.05 27.18
28.93
31.53 32.05
33.35
35.37
36.69
37.95
40.33
Fig. 4. GC-MS Spectra of crude lecithin
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2ppm 200 18 0 160 1 40 120 100
34 32 30 28 26 24 22 20 18 16 14 12 ppm
80 60 40 20 0
ppm
(a) (b)
Fig. 5. NMR spectra’s of crude lecithin (a) (1H NMR) (b) (13C NMR)
ppm revealed the presence of inorganic orthophosphate and
monoester phosphate groups present in the sample9,12. It also
elaborated various structural elucidations of compounds
present in them.
Thermal analysis by DSC-TGA: Analysis of crude
lecithin using DSC-TGA techniques elaborates the thermal
properties of the sample (Fig. 6). DSC spectral peaks at 75.24,
274.85 and 302.53 ºC show the glass transition temperature,
recristallization point and melting point of the crude lecithin
sample, respectively. The recorded thermogravimetric spectra
showed two distinct degradation points, which confirmed
coexistence of more than one degradation process. At lowest
temperature of around 80 ºC it showed the breakage of water
3738 Balaguru et al. Asian J. Chem.

120
100
80
60
40
20
0
W
e
i
g
h
t
(
%
)
Temperature (°C)
100 200 300 400 500
Heat flow (mW)
-6
-8
-10
-12
-14
-16
-18
-20
0
Exo Up
75.24°C
Residual
15.12%
(1.286 mg)
305.53°C
274.85°C
Fig. 6. DSC-TGA curve of crude lecithin
linkages and at higher temperature of 400 ºC showed the com-
plete degradation of the crude lecithin compound11-13.
Fourier transform infrared spectroscopy: FTIR spec-
troscopy using ATR technique was done and the spectral curve
was obtained for crude lecithin sample. Fig. 7 presents various
bands of spectra obtained in between the range of 4000-400
cm-1 which were due to different chemical bond stretching and
bending of molecules in the sample. Fig. 7 shows the FTIR
spectra of crude lecithin and their characteristic functional
groups stretching were elaborated in Table-2.
100
90
80
70
60
50
40
30
20
10
0
T
r
a
n
s
m
i
t
t
a
n
c
e
(
%
)
4000
3000
2000
1500
1000
400
Wavelength (cm )
–1
3431.16
2921.62
2852.65
2359.32
2322.15
3865.43
3976.05
1803.29
2080.17
1734.67
1632.27
1460.21
1376.05
1224.351158.72
689.00
576.74
530.79
1056.59
770.19
Fig. 7. FTIR spectra of quail egg lecithin crude
TABLE-2
FUNCTIONAL GROUPS PRESENT IN CRUDE LECITHIN
AND THEIR MOLECULAR STRETCHING
Functional groups Molecules Wave number (cm-1)
Alkyl C-H Stretch 2322.15
Aldehydes =C-H Stretch
C=O Stretch
2852.65
1734.67
Esters C- O Stretch 1056.59
1124.35
Carbonyl C=O Stretch 1803.29
Alkenes C=C Stretch
=C-H Bending
1632.27
770.19
Alkanes -C-H Stretch 2852.65
1376.05
Acid -O-H Stretch
C-O Stretch
2921.62
1224.35
Amide N-H Stretch 3431.16
Aromatic C=C Stretch 1460.21
Cytotoxicity study of lecithin: Extracted crude lecithin
from quail’s egg was evaluated for cytotoxic potentials in normal
cell line (NIH3T3) using MTT assay techniques (Figs. 8 and
9). In vitro studies were carried out using various concentra-
tions such as 12.5, 25, 50, 100 and 200 µg/mL and the percen-
tage of cell viability observed was in the range of 98.44, 97.80,
92.30, 85.07 and 79.12, respectively. From the data obtained
it is concluded that lecithin at a higher concentration of 200
µg/mL showed less cytotoxicity in normal cell line. Lecithin
as a biocompatible material can be utilized in cosmet ics and
in other pharmaceutical preparations and in developing non
toxic human friendly products.
100
80
60
40
20
0
%
C
e
l
l
v
i
a
b
i
l
i
t
y
12.5 25 50 100 200
Concentration (µg/mL)
Fig. 8. Percentage of cell viability against lecithin at different concentrations
Fig. 9. NIH3T3 cell line treated with Quails egg lecithin. (a) control (b) 12.5 µL/mL (c) 200 µL/mL
Vol. 26, No. 12 (2014) Identification and Exploration of Lecithin from Novel Sources and its Applications 3739

Conclusion
Lecithin was successfully isolated from quail egg and
analyzed both quantitatively and qualitatively by well estab-
lished techniques like TLC, GC-MS, NMR, DSC-TGA, FTIR
and also evaluated for cytotoxic potential. The data of the results
were encouraging and depicted that the isolated compound
contains fatty acids and esters in them. NMR and FTIR also
confirmed the presence of these components. Present study
inferred that it can be used as biocompatible material for various
applications in pharmaceutical industry and in cosmetic indus-
tries for developing creams and gels.
ACKNOWLEDGEMENTS
The authors are thankful to the management of SASTRA
University, Thanjavur for providing the necessary facilities to
carry out the project.
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