Spectral analysis and anti-bacterial activity of methanolic fruit extract of Citrullus colocynthis using gas chromatography-mass spectrometry

Phytochemicals are chemical compounds often referred to as secondary metabolites. Thirty three bioactive phytochemical compounds were identified in the methanolic extract of Citrullus colocynthis. The identification of phytochemical compounds is based on the peak area, retention time molecular weight and molecular formula. Gas chromatography-mass spectrometry (GC-MS) analysis of C. colocynthis revealed the existence of the methyl 6-oxoheptanoate, hexanoic acid, 2-isopropyl-2-methyl5-oxo-, methyl ester, dodecanoic acid, 3-hydroxy, benzofuran,2,3-dihydro, 1,1-Cyclopropanedimethanol, 2-methyl-α-phenyl, 1,1-cyclopropanedimethanol, 2-methyl-α-phenyl, 12,15-octadecadiynoic acid, methyl ester, (5ß)pregnane-3,20ß-diol, 14α,18α-[4-methyl-3-oxo-(1-oxa-4-azabutan, 3-(N,Ndimethyllaurylammonio)propanesulfonate, 2H-1-benzopyran-3,4-diol,2-(3,4-dimethoxyphenyl)3,4dihydro-6-met, 11,13-dihydroxy-tetradec-5-ynoic acid, methyl ester, Cyclopenta[1,3]cyclopropa[1,2]cycloheptan-3(3aH)-one,1,2,3b,6,7, 4-(2,4,4-trimethyl-cyclohexa-1,5dienyl)-but-3-en-2-one, 1-tetradecanamine,N,N-dimethyl, α-D-glucopyranoside, O-α-D-glucopyranosyl(1,fwdarw.3)-ß-D-fructo, acetamide, N-methyl-N-[4-(3-hydroxypyrrolidinyl)-2-butynyl]-, 9octadecenamide,(z)-, butyrophenone,2 ́,3,4 ́,6 ́-tetramethyl-, ethyl 5,8,11,14,-eicosatetraenoate, 9,12,15octadecatrienoic acid, 2,3,-dihydroxypropyl ester, (Z,Z,Z)-, 1H-cyclopropa[3,4]benz[1,2-e]ezulene 5,7b,9,9a 476.241018tetrol,1a,1b,4,4a, 9,12,15-octadecatrienoic acid, 9,10-Secocholesta -5,7,10(19)triene-3,24,25,-triol,(3ß,5Z,7E)-, 9,12,15-Octadecatrienoic acid,2,3-dihydroxypropyl ester, (Z,Z,Z)-, triazido-(1,2,3,4,5-pentamethylcyclopenta-2,4-dienyl)-german, ethyl iso-allocholate, α-N-Normethadol, Octadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester, phthalic acid , decyl oct-3-yl ester, 1,2Benzenedicarboxylic acid , bis(8-methylnonyl)ester, phthalic acid, di(6-ethyl-3-octyl)ester, y-tocopherol, 1,4-ethanonaphthalene -6,9(4H)-dione,1,4a,5,8a-tetrahydro-4,5,7,10 and vitamin E. Methanolic extract of bioactive compounds of C. colocynthis was assayed for in vitro antibacterial activity against Proteus mirabilis, Escherichia coli, Pseudomonas aerogenosa, P. mirabilis, Staphylococcus aureus and Klebsiella pneumonia by using the diffusion method in agar. The zone of inhibition was compared with different standard antibiotics. The diameters of inhibition zones ranged from 4.91±0.260 to 1.03±0.200 mm for all treatments.


INTRODUCTION
Secondary metabolites play an important role as source of new compounds in pharmaceutical industry and need special consideration.Biotechnological approaches, specifically plant tissue culture plays a vital role in search for alternatives to production of desirable medicinal compounds from plants (Lamson and Brignall, 2000;Rao and Ravishankar, 2002;Rusak et al., 2002;Scalbert et al., 2005;Karuppusamy, 2009).Citrullus colocynthis is the plant which produces colocynth apples and is very similar to the common watermelon vine.The colocynth apples are small fruit pulp that taste bitter.The plant bears solitary sterile flowers and branched tendrils.It has a large, fleshy perennial root, which sends out slender, tough, angular, scabrid vine-like stems.The leaves are angular, lobed and, as already stated.C. colocynthis (L.) Schard. is an Iranian medicinal plant that has traditionally been used as an abortifacient and to treat constipation, oedema, bacterial infections, cancer and diabetes (Delazar et al., 2006).The capacity for plant cell, tissue and organ cultures to produce and accumulate many of the same valuable chemical compounds as the parent plant in nature, has been recognized almost since the inception of in vitro technology.The strong and growing demand in today's market place for natural, renewable products has refocused attention on in vitro plant materials as potential factories for secondary phytochemical products (Karuppusamy, 2009;Al-Tameme et al., 2015a;Hameed et al., 2015a).Colocynthin is soluble in water and alcohol, but insoluble in benzol, benzin, carbon disulfide and ether.Dilute acids resolve it into dextrose and tasteless colocynthein, acetic acid being likewise formed (Delazar et al., 2006).Walz obtained from an alcoholic extract of colocynth, an ethersoluble crystalline and tasteless substance insoluble in water, which he called colocynthin (Memon et al., 2003).

Plant and extraction
Fresh plants were collected from local market in Hilla city, middle of Iraq.The plant was identified by Prof. Dr. Abdul-Kareem, Babylon University, Faculty of Science for Women.Fruits were thoroughly washed using deionized water, mopped with tissue paper and airdried in a shade to prevent the decomposition of chemical constituents (Al-Tameme et al., 2015b;Hameed et al., 2015b).All seeds were separated manually from the pulp.The dried pulp of fruits was homogenized with a grinder (Muleinex) to fine powder before extraction.The pulp powder was extracted three times at room temperature with 100 ml of methanol for 6 h according to Halliwell and Gutteridge (1985) and Yoshikawa et al. (2007).Methanol-soluble portions were pooled from the 300 ml filtrate.The oven (45 to 50°C) dried ethanol extract (10 g) was dissolved in freshly prepared normal saline (0.9%) to a final stock solution (10 mg/ml).

Gas chromatography-mass spectrum (GC-MS) analysis
The GC-MS analysis of the plant extract was made in a 7890 Agilent A instrument under computer control at 70 eV.About 1 μl of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 45 min (Mohammed and Imad, 2013;Kareem et al., 2015).As the compounds were separated, they eluted from the column and entered a detector which was capable of creating an electronic signal whenever a compound was detected.The greater the concentration in the sample, the bigger the signal obtained which was then processed by a computer.The time from when the injection was made (Initial time) to when elution occurred is referred to as the retention time (RT).While the instrument was run, the computer generated a graph from the signal called chromatogram.Each of the peaks in the chromatogram represented the signal created when a compound eluted from the gas chromatography column into the detector (Imad et al., 2014a).The X-axis showed the RT and the Yaxis measured the intensity of the signal to quantify the component in the sample injected.As individual compounds eluted from the Gas chromatographic column, they entered the electron ionization (mass spectroscopy) detector, where they were bombarded with a stream of electrons causing them to break apart into fragments.The fragments obtained were actually charged ions with a certain mass (Imad et al., 2014b).The mass/charge (M/Z) ratio obtained was calibrated from the graph obtained, which was called as the mass spectrum graph which is the fingerprint of a molecule.Before analyzing the extract using GC-MS, the temperature of the oven, the flow rate of the gas used and the electron gun were programmed initially.The temperature of the oven was maintained at 100°C.Helium gas was used as a carrier as well as an eluent.The flow rate of helium was set to 1 ml/min.The electron gun of mass detector liberated electrons having energy of about 70eV.The column employed here for the separation of components was Elite 1 (100% dimethyl poly siloxane).The identity of the components in the extracts was assigned by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with published literatures.Compounds were identified by comparing their spectra to those of the Wiley and NIST/EPA/NIH mass spectral libraries (Imad et al., 2014c;Hamza et al., 2015).

Determination of antibacterial activity of crude bioactive compounds of C. colocynthis
The test pathogens (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) were swabbed in Muller Hinton agar plates.60 μl of the plant extract was loaded on the bored wells (Hameed et al., 2015c;Hussein et al., 2015).The wells were bored in 0.5 cm in diameter.The plates were incubated at 37°C for 24 h and were examined.After the incubation, the diameter of inhibition zones around the discs was measured.

Statistical analysis
Data were analyzed using analysis of variance (ANOVA) and differences among the means were determined for significance at P < 0.05 using Duncan's multiple range test (by SPSS software) Version 9.1 (Jasim et al., 2015).

Figure 2 .
Figure 2. Structure of methyl 6-oxoheptanoate present in the Citrullus colocynthis by using GC-MS analysis

Figure 4 .
Figure 4. Structure of dodecanoic acid, 3-hydroxy present in the Citrullus colocynthis by using GC-MS analysis.

Figure 25 .
Figure 25.Structure of ethyl iso-allocholate present in the Citrullus colocynthis by using GC-MS analysis.

Figure 26 .
Figure 26.Structure of α-N-normethadol present in the Citrullus colocynthis by using GC-MS analysis.

Figure 28 .
Figure 28.Structure of phthalic acid, decyl oct-3-yl ester present in the Citrullus colocynthis by using GC-MS analysis.

Figure 31 .
Figure 31.Structure of γ-tocopherol present in the Citrullus colocynthis by using GC-MS analysis.

Figure 33 .
Figure 33.Structure of vitamin E present in the Citrullus colocynthis by using GC-MS analysis.

Figure 34 .
Figure 34.Structure of octadecanoic acid present in the Citrullus colocynthis by using GC-MS analysis.

Table 1 .
Major phytochemical compounds identified in methanolic extract of Citrullus colocynthis.

Table 2 .
Zone of inhibition (mm) of test bacterial strains to Citrullus colocynthis bioactive compounds and standard antibiotics. .colocynthis possesses remarkable antimicrobial activity, which is mainly due to α-Dglucopyranoside, O-α-D-glucopyranosyl, phthalic acid and y-tocopherol.According to these findings, it could be said that the methanolic extract act as antibacterial agents.