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Sharangouda et al.
Research Article
Article Received on
5 November 2013,
Revised on 02 December
2013,
Accepted on 05 January
2014
ABSTRACT
The aim of the current study was to investigate the phytochemical and
GCMS analysis of petroleum ether extract of Citrus medica seeds. The
bioactive constituents were analysed by phytochemical (qualitative)
and GCMS method. Preliminary studies showed the presence of
*Correspondence for
Author:
Dr. Sharangouda J. Patil
Research Associate
high concentration of Oleic acid with Retention Time 18.57 has peak
18.51 has peak area 11.51%, -Sitosterol with Retention Time 30.30
has peak area 11.17 % and Hexadecanoic acid with Retention Time
16.46 has peak area 10.35 %. The presence of some of these bioactive
constituents in the plant extract may provide the
scientific evidences for the antifertility activity and contraceptive properties of the plant.
Key words: Citrus medica, GCMS analysis, Bioactive constituents, Antifertility,
Contraceptive.
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INTRODUCTION
The natural product study is gaining much importance in recent years due to wide
applications of bioactive molecules of the medicinal plants and their products. Different
strategies have been developed for the selection of particular plant for the study. The plants
selected with different strategies are extracted with different organic solvents with increased
polarity. The plant extracts are screened for the activity of interest. The active extract is
subjected to isolation of active constituent(s) present in that with different analytical
techniques. The analogues of isolated molecules are characterized and structural modification
has been done to enhance the desired activity and minimize the unwanted side effects.
In this context, Citrus medica seeds were selected for the study to find out their novel
bioactive constituents. Citrus medica (Rutaceae) is an indigenous small tree or shrub. Its
seeds are as in the orange, but smaller upto 12-15 seeded in a one fruit. The seeds are
indigestible, heavy, heating to the body, stimulus, toxic, good for piles and in biliousness,
cure inflammations and Kapha (Ayurvedic) [1-2].
described that petroleum ether extract of C. medica Linn. seeds (200 and 400 mg/kg) induced
significant antidiabetic, hypocholesterolemic, hypolipidemic activity and in my previous
findings of petroleum
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analysed in GCMS for different constituents. Using computer searches on a NIST REFPROP
Version 9.1 database and comparing the spectrum obtained through GCMS compounds
present in the plants sample were identified.
Identification of Bioactive Constituents
Interpretation on Mass-Spectrum GCMS was carried out by using the database of National
institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of
the unknown components was compared with the spectrum of known components stored in
the NIST library. The name, molecular formula, weight and chemical structure of the
components of the test materials were ascertained.
RESULTS AND DISCUSSION
Phytochemical Analysis
The petroleum ether extract showed positive test for alkaloids, glycosides, flavones, proteins,
amino acids, fats and oils.
GCMS Analysis (Table 1 &2; Fig. 1)
GCMS chromatogram of the petroleum ether extract of Citrus medica seeds (Fig. 1) showed
23 peaks indicating the presence of twenty three bioactive constituents. The active principles
with their retention time (RT), molecular formula, molecular weight (MW), concentration
(peak area %) and chemical structures were presented in Table 1& 2. The total numbers of
compounds identified in petroleum ether extract were the GCMS retention time (RT) and
percentage peak of the individual compounds. The results revealed that Oleic acid (23.27 %),
9,12-Octadecadienoic acid (11.51 %), -Sitosterol (11.17 %) and Hexadecanoic acid (10.35
%) were found as the 4 major constituents covering higher concentration of area in the
petroleum ether extract. The three constituents such as Octadecanoic acid (6.65%),
Hexadecanoic acid, trimethylsilyl ester (5.00 %) and Diethyl phthalate (4.37 %) covered
moderate concentration of area and remaining 16 minor constituents shown below (2.79 to
0.44 %) of the concentration in the petroleum ether extract of the Citrus medica seeds.
The study of GCMS data suggests that, possible presence of unsaturated hydroxy fatty acids
in the sample under investigation. From the above data one can anticipate presence of
mixture of hexadeconic acid, 9,12-octa dedeconic acid, -Sitosterol and oleic acid in the
structure of these molecule are in accordance with the proposal made further possible mixture
of fatty acids were present in the isolated sample.
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El-Alfy [12], described that GCMS analysis of Citrus medica leaves possessing both
saponifiable and unsaponifiable matters revealed the presence of thirty three components (28
hydrocarbons and 5 sterols) in the unsaponifiable fraction, the major hydrocarbon was nHeneicosane (16.7%) while the major sterol was -Sitosterol (4.03%) and 15 components in
the saponifiable matter it's major component was hexadecanoic acid (19.93%). Similar
studies have shown that unsaturated fatty acids, but not saturated fatty acids, modulate
estrogen and/or ER(s) by alterations in estradiol binding to receptors and/or by cleaving
native ER(s) [13]. Phytoestrogens are of biological interest because they exhibit oestrogenic
activity, both in vitro and in vivo, by weakly binding to oestrogen receptors [14].
Nonesterified fatty acids may influence cell growth and proliferation by modifying
membrane fluidity [15]. 10-hydroxy-trans-2-decenoic acid, 10- hydroxydecanoic acid, trans2-decenoic acid and 24-methylenecholesterol were reported to induce mild hypertrophy of
the luminal epithelium of the uterus, but were not associated with an increase in uterine
weight.
.
Table 1. Bioactive constituents peak number, name, retention time and peak area (%)
of seed extract of Citrus medica by GCMS analysis
Peak No
Retention Time
2-Heptenal
2.65
2.31
2=-Octenal, (E)-
6.45
1.18
2, 4-Hexadienal, (E,E)-
6.98
1.77
2, 4-Decadienal
7.35
2.22
1, 2-Benzenedicarboxylic acid,
15.34
0.84
Hexadecanoic acid
16.46
10.35
Diethyl Phthalate
16.52
4.37
17.45
5.00
ester
9
18.51
11.51
10
Oleic acid
18.57
23.27
11
Octadecanoic acid
18.80
6.65
12
Dotriacontane
21.28
0.44
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13
Hexatriacontance
22.28
1.18
14
1,2-Benzenedicarboxylic acid, 3-
22.84
0.69
nitro15
Nonadecane
23.25
1.81
16
Heneicosane
24.17
2.30
17
Tetracosane
25.07
2.34
18
Hexatriacontane
25.94
2.79
19
Pentadecane
26.78
2.53
20
Tritetracontane
27.62
2.11
21
Tetratetracontane
28.57
1.42
22
Octadecane,1-chloro-
29.65
1.73
23
-Sitosterol
30.30
11.17
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Table 2. Bioactive constituents name, molecular formula, molecular weight and chemical structure of seed extract of Citrus medica by
GCMS analysis
Name
of
the Molecular
Molecular
Constituents
Formula
Weight
2-Heptenal
C7H12O
112.1696
Chemical Structure
(E)
2=-Octenal, (E)2,
C8H14O
126.1962
4-Hexadienal, C6H8O
(E,E)-
96.1241
2, 4-Decadienal
1,
C10H16O
(E)
152.2334
(E)
(E)
( E)
2- C16H22O4
Benzenedicarboxyl
O
O
O
methylpropyl)
ester
278.3435
Hexadecanoic acid
C16H32O2
256.4241
O
OH
Diethyl Phthalate
C12H14O4
222.2372
O
O
O O
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Hexadecanoic acid, C19H40O2Si
trimethylsilyl ester
9,
12- C18H32O2
280.4455
O
octadecadienoic
OH
acid (Z,Z)
Oleic acid
C18H34O2
282.4614
HO
O
Octadecanoic acid
C18H36O2
284.4772
Dotriacontane
C32H66
450.8664
Hexatriacontance
C36H74
506.9728
1,2-
C8H5NO6
211.1284
Benzenedicarboxyl
ic acid, 3-nitro-
OH
OO N+
O
HO
HO
O
Nonadecane
C19H40
268.5209
Heneicosane
C21H44
296.5741
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Tetracosane
C24H50
338.6538
Hexatriacontane
C36H74
506.9728
Pentadecane
C15H32
212.4146
Tritetracontane
C43H88
605.1588
Tetratetracontane
C44H90
619.1854
Octadecane,1-
C18H37Cl
288.939
C29H50O
414.7067
Cl
chloro-Sitosterol
HO
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CONCLUSION
In the present study 23 bioative constituents have been identified from petroleum ether
extract of Citrus medica seeds by Gas Chromatography and Mass Spectrometry (GCMS)
analysis. The presence of various bioactive compounds justifies the use of whole plant
various ailments by traditional practitioners. The above observations suggest the C. medica
seeds are rich in fatty acid and can be utilized for the commercial application as
contraceptive.
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