GCMS ANALYSIS OF BIOACTIVE CONSTITUENTS FROM THE PETROLEUM ETHER EXTRACT OF CITRUS MEDICA SEEDS

www.wjpps.com Vol 3, Issue 2, 2014.

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Sharangouda et al. World Journal of Pharmacy and Pharmaceutical Sciences

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

1Department of Post-Graduate Studies and Research in Zoology, Gulbarga University,

Gulbarga-585106, Karnataka, India 2Department of Biology, Vishwa Jyothi P.U. College, Siraguppa - 583121, Bellary,

Karnataka, India. 3Department of Microbiology, Maharani Science College for Women’s, Bangalore - 560001,

Karnataka, India 4Department of Zoology, L.V.D. College, Raichur - 584103, Karnataka, India.

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

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

high concentration of Oleic acid with Retention Time 18.57 has peak

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

18.51 has peak area 11.51%, ß-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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Article Received on 5 November 2013, Revised on 02 December 2013, Accepted on 05 January 2014

*Correspondence for

Author:

Dr. Sharangouda J. Patil

Research Associate

NAIP Livelihood Project

National Institute of Animal

Nutrition and Physiology

(NIANP & ICAR), Adugodi,

Bangalore, India.


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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

(28-30°C).


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Soxhlet Extraction of Seed Constituents

The shade dried, powdered 100gm seed material was soxhleted with petroleum ether (b.p. 60-

80°C) 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 (50-60°C) 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 4°C 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

2µl 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 AOC-

20i 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 250°C; ion-source temperature 280°C. The oven temperature was programmed

from 110°C (isothermal for 2 min), with an increase of 10°C/min, to 200°C/min, then

5°C/min to 280°C/min, ending with a 9 min isothermal at 280°C. 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 %), ß-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, ß-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 n-

Heneicosane (16.7%) while the major sterol was β-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, trans-

2-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 (%)

1 2-Heptenal 2.65 2.31

2 2=-Octenal, (E)- 6.45 1.18

3 2, 4-Hexadienal, (E,E)- 6.98 1.77

4 2, 4-Decadienal 7.35 2.22

5 1, 2-Benzenedicarboxylic acid,

bis (2-methylpropyl) ester

15.34 0.84

6 Hexadecanoic acid 16.46 10.35

7 Diethyl Phthalate 16.52 4.37

8 Hexadecanoic acid, trimethylsilyl

ester

17.45 5.00

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-

nitro-

22.84 0.69

15 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 β-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

Constituents

Molecular

Formula

Molecular

Weight

Chemical Structure

2-Heptenal C7H12O 112.1696 (E)

O 2=-Octenal, (E)- C8H14O 126.1962 O 2, 4-Hexadienal,

(E,E)-

C6H8O

96.1241

(E) (E)O

2, 4-Decadienal C10H16O 152.2334 (E) ( E) O 1, 2-

Benzenedicarboxyl

ic acid, bis (2-

methylpropyl)

ester

C16H22O4

278.3435

O

OOO

Hexadecanoic acid C16H32O2 256.4241 O

OH Diethyl Phthalate C12H14O4 222.2372

OOOO


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Hexadecanoic acid,

trimethylsilyl ester

C19H40O2Si 328.6052 O

OSi

9, 12-

octadecadienoic

acid (Z,Z)

C18H32O2 280.4455 O

OH

Oleic acid C18H34O2 282.4614 HO

O

Octadecanoic acid C18H36O2 284.4772 O OH

Dotriacontane C32H66 450.8664

Hexatriacontance C36H74 506.9728 1,2-

Benzenedicarboxyl

ic acid, 3-nitro-

C8H5NO6 211.1284

OHO

O

HO

N+OO-

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-

chloro-

C18H37Cl 288.939 Cl

β-Sitosterol C29H50O 414.7067

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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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.

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medica seeds extract on female Albino Mice. Glob J Pharma Technol, 2011; 3: 14-21.

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12. El-Alfy TS, Hetta MH, Yassin NZ, Rahman RFA, Kadry EM. Estrogenic activity of

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GCMS ANALYSIS OF BIOACTIVE CONSTITUENTS FROM THE PETROLEUM ETHER EXTRACT OF CITRUS MEDICA SEEDS

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