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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Sharangouda et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 2, 1239-1249.

Research Article

ISSN 2278 4357

GCMS ANALYSIS OF BIOACTIVE CONSTITUENTS FROM THE


PETROLEUM ETHER EXTRACT OF CITRUS MEDICA SEEDS
Sharangouda J. Patil1*, Venkatesh S.2, Vishwanatha T.3, Sneha R. Banagar4,
Ravikumar J. Banagar4 and Saraswati B. Patil1
1

Department of Post-Graduate Studies and Research in Zoology, Gulbarga University,


Gulbarga-585106, Karnataka, India
2

Department of Biology, Vishwa Jyothi P.U. College, Siraguppa - 583121, Bellary,


Karnataka, India.

Department of Microbiology, Maharani Science College for Womens, Bangalore - 560001,


Karnataka, India
4

Department of Zoology, L.V.D. College, Raichur - 584103, Karnataka, India.

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

alkaloids, glycosides, flavones, proteins, amino acids, fats and oils. In


the GCMS analysis, 23 bioactive constituents were identified in the
petroleum ether extract. The identification of these constituents are in

NAIP Livelihood Project

high concentration of Oleic acid with Retention Time 18.57 has peak

National Institute of Animal

area 23.27%, 9, 12- Octadecadienoic acid (Z,Z) with Retention Time

Nutrition and Physiology

18.51 has peak area 11.51%, -Sitosterol with Retention Time 30.30

(NIANP & ICAR), Adugodi,


Bangalore, India.

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].

The studies of Archana et al,[3]

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

ether crude extract of C. medica seeds investigated their

antiimplantation, estrogenic, antiovulatory, abortifacient activities [4-8] and toxicity studies


[9-10] in rats and mice. But, there is no much reports on the detailed analysis of GCMS and
bioactive constituents of this plant material, hence, it was planned to take up detailed
investigation on Citrus medica seeds for isolation of active biomolecules and its
pharmacological activities from the potent constituent.
MATERIALS AND METHODS
Collection of Seeds
The fresh seeds of Citrus medica were collected from Hyderabad Karnataka areas of northern
region of Karnataka, during fruiting season i.e., in the month of July to October and
authenticated at the herbarium, Department of Botany, Gulbarga University, Gulbarga,
Karnataka, India. Collected seeds material was immediately sprayed with ethanol to cause the
enzymatic degradation of secondary metabolites. The seeds were shade dried, chopped into
small fragments and powdered inside the laboratory within 10-15 days at room temperature
(28-30C).

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Soxhlet Extraction of Seed Constituents


The shade dried, powdered 100gm seed material was soxhleted with petroleum ether (b.p. 6080C) in a soxhlet extractor for 48 hours. The extract was concentrated to dryness in a flash
evaporator (Buchi) under reduced pressure and con-trolled temperature (50-60C) to obtain
the crude extract. Remaining trace of the solvent if any was further removed by placing the
crude extract in vaccum overnight. The yield is 15gm and yellow oily coloured extract was
obtained. The extract was stored in refrigerator at 4C until used for experiment.
Phytochemical Analysis
Phytochemical analysis of the petroleum ether extract of the plant was carried out in order to
know the class of organic compounds present in the different extracts of the seeds selected
for the study, which further facilitates for the identification of active constituents and their
isolation.
The petroleum ether extract of Citrus medica seeds were subjected to standard chemical tests
as described by Harnborne, [11] to determine the presence (qualitatively) or absence of
alkaloids, steroids, glycosides, saponins, flavones, carbohydrates, proteins and amino acids,
fats and oils and phenols.
Gas Chromatography Mass Spectroscopy Analysis
Preparation of Extract
2l of the petroleum ether extract of Citrus medica seeds was employed for GCMS analysis.
Instruments and Chromatographic Conditions
GCMS analysis was carried out on a GC Clarus 500 Perkin Elmer system comprising a AOC20i autosampler and gas chromatograph interfaced to a mass spectrometer (GCMS)
instrument employing the following conditions: column Elite-1 fused silica capillary column
(30 0.25 mm ID 1EM df, composed of 100% Dimethyl poly siloxane), operating in
electron impact mode at 70 eV; helium (99.999%) was used as carrier gas at a constant flow
of 1ml/min and an injection volume of 0.5 EI was employed (split ratio of 10:1) injector
temperature 250C; ion-source temperature 280C. The oven temperature was programmed
from 110C (isothermal for 2 min), with an increase of 10C/min, to 200C/min, then
5C/min to 280C/min, ending with a 9 min isothermal at 280C. Mass spectra were taken at
70 eV; a scan interval of 0.5 s and fragments from 40 to 550 Da. The plant extract was
dissolved in methanol and filtered with polymeric solid phase extraction (SPE) column and

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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

Name of the Constituents

Retention Time

Peak Area (%)

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

bis (2-methylpropyl) ester


6

Hexadecanoic acid

16.46

10.35

Diethyl Phthalate

16.52

4.37

Hexadecanoic acid, trimethylsilyl

17.45

5.00

ester
9

9, 12-octadecadienoic acid (Z,Z)

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

Fig. 1: GCMS Spectrum of Petroleum ether extract of Citrus medica seeds

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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

ic acid, bis (2-

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

World Journal of Pharmacy and Pharmaceutical Sciences


328.6052
Si

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.
REFERENCES
1. Kirtikar KR, Basu AD. In: Indian Medicinal Plants. Vol-I, Lalit Mohan Basu, Allahabad,
1935, 53, 1575.
2. Nadkarni AK, Nadkarni KM. Indian Medicinal plants. Vol-II, Popular Book Depot,
Bombay, 1976.
3. Archana N, Amit Joshi, Vijay Juyal, Tirath Kumar. Antidiabetic and Hypolipidemic
activity of Citrus medica Linn. seed extract in Streptozotocin induced Diabetic rats.
Pharmacognosy J, 2011; 23, 12.
4. Sharangouda, Patil SB. Phytochemical screening and antifertility activity of various
extracts of Citrus medica (Lemon) seeds in albino rats. Adv Pharm Tox, 2007; 2: 71-74.
5. Sharangouda, Patil SB. Post-coital antifertility activity of Citrus medica seeds. The
Bioscan, 2007; 2: 305-310.
6. Sharangouda, Patil SB. Estrogenic activity of petroleum ether extract of seed of Citrus
medica on immature albino rats. Int J Green Pharmacy, 2008; 2: 91-94.
7. Sharangouda, Patil SB. Efficacy of Citrus medica seeds extracts on reproductive activities
in female albino rats. Pharmacologyonline, 2009; 2: 803-817.
8. Sharangouda,

Patil

SB.

Antifertility

principles

from

Citrus

medica

seeds:

pharmacological and phytochemical studies. Asian J Exp Sci, 2009; 24: 155-161.
9. Patil SJ, Patil SB. Pre-clinical toxicity studies of orally administered petroleum ether
extract of Citrus medica seeds on the reproductive organs of female mice. Int J Contemp
Res Rev, 2010; 1: 1-6.
10. Patil SJ, Patil SB. Toxicity studies on Hepatic, Nephric and Endocrine Organs of Citrus
medica seeds extract on female Albino Mice. Glob J Pharma Technol, 2011; 3: 14-21.
11. Harnborne JB. Phytochemical Methods Chapman and Hall Ltd. New York, 1973; pp.37214.

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12. El-Alfy TS, Hetta MH, Yassin NZ, Rahman RFA, Kadry EM. Estrogenic activity of
Citrus medica L. leaves growing in Egypt. J App Pharma Sci, 2012; 02 (08): 180-185.
13. Borras M, Leclercq G. Modulatory effect of nonesterified fatty acids on structure and
binding characteristics of estrogen receptor from MCF-7 human breast cancer cells. J
Recept Res, 1992; 12: 46384.
14. Adlercreutz H. Diet, breast cancer and sex hormone metabolism. Ann N Y Acad Sci.
1990; 595: 281-290.
15. Urns CP, Luttenegger DG, Dudley DT, Buettner GR, Spector AA. Effect of modification
of plasma membrane fatty acid composition on fluidity and methotrexate transport in
L1210 murine leukemia cells. Cancer Res, 1979; 39: 1726 32.

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