1 Vol. 14(46), pp , 18 November, 2015 DOI: /AJB Article Number: 525E ISSN Copyright 2015 Author(s) retain the copyright of this article African Journal of Biotechnology Full Length Research Paper Spectral analysis and anti-bacterial activity of methanolic fruit extract of Citrullus colocynthis using gas chromatography-mass spectrometry Salah Ali Idan, Ali Hussein Al-Marzoqi and Imad Hadi Hameed* Department of Biology, Babylon University, Hilla City, Iraq. Received 1 September, 2015; Accepted 5 November, 2015 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-methyl- 5-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-1,5- dienyl)-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]-, 9- octadecenamide,(z)-, butyrophenone,2,3,4,6 -tetramethyl-, ethyl 5,8,11,14,-eicosatetraenoate, 9,12,15- octadecatrienoic acid, 2,3,-dihydroxypropyl ester, (Z,Z,Z)-, 1H-cyclopropa[3,4]benz[1,2-e]ezulene - 5,7b,9,9a tetrol,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, 1,2- Benzenedicarboxylic 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. Key words: Gas chromatography-mass spectrometry, phytochemicals, Citrullus colocynthis.
2 3132 Afr. J. Biotechnol. 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). MATERIALS AND METHODS 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 Y- axis 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. *Corresponding author. Tel: Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License
3 Idan et al Table 1. Major phytochemical compounds identified in methanolic extract of Citrullus colocynthis. S/N Phytochemical compound RT (min) Formula Molecular weight Exact mass Chemical structure MS Fragment-ions Pharmacological actions 1 Methyl 6-oxoheptanoate C8H14O ,84,111,126,143 Anti-cancer activity 2 Hexanoic acid, 2-isopropyl-2-methyl-5-oxo-, methyl ester C11H20O ,69,83,98,115,130,157,1 68,185 Antifungal activity 3 Dodecanoic acid, 3-hydroxy C12H24O ,69,83,96,112,123,138,1 51,180,200 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). RESULTS AND DISCUSSION Herbal medicine represents one of the most important fields of traditional medicine all over the world. Different extracts from traditional medicinal plants have been tested to identify the source of the therapeutic effects (Cragg et al., 1997; Hameed et al., 2015d). GC-MS analysis of the compounds carried out in methanolic extract of C. colocynthis is shown in Table 1. The GC-MS chromatogram of the 33 peaks of the compounds detected are as shown in Figure 1. Chromatogram GC-MS analysis of the methanol extract of C. colocynthis showed the presence of twenty major peaks and the components corresponding to the peaks were determined as follows: the first set up peak was determined to be methyl 6- oxoheptanoate (Figure 2). The second peak was indicated to be hexanoic acid, 2-isopropyl-2- methyl-5-oxo-, and methyl ester (Figure 3). The next peaks were considered to be 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,N- Dimethyllaurylammonio) 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-trimethylcyclohexa-1,5-dienyl)- 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]-, 9-
7 Idan et al Table 1. Contd. 17 Butyrophenone,2,3,4,6 -tetramethyl C14H20O ,91,119,147,171, Ethyl 5,8,11,14,-eicosatetraenoate C22H36O ,67,79,91,119,150,175,2 17,275, ,12,15-Octadecatrienoic acid, 2,3,- dihydroxypropyl ester, ( Z,Z,Z) C21H36O ,67,79,95,109,155,173,2 32,261,291,321,352 Anti-inflammatory and CNS depressant activity 20 1H-Cyclopropa[3,4]benz[1,2-e]ezulene - 5,7b,9,9a tetrol,1a,1b,4,4a C26H36O ,69,131,200,296,324,35 6,416,476 acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester, phthalic acid, decyl oct-3-yl ester, 1,2- Benzenedicarboxylic 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 (Figures 4 to 34). In this study, five clinical pathogens were selected for antibacterial activity, namely, S. aureus, K. pneumoniae, P. aeroginosa, E. coli, and Proteus mirabilis. The maximum zone formation against P. aeroginosa was from 4.91±0.260 to 1.03±0.200 (Table 2).
8 3138 Afr. J. Biotechnol. Table 1. Contd. 21 9,12,15-Octadecatrienoic acid C18H30O ,67,79,93,108,121,135,2 22,249,278 Anti-inflammatory anti-atherogenic properties and 22 9,10-Secocholesta triol,(3ß,5z,7e)- -5,7,10(19)-triene-3,24,25, C18H36O ,69,91,118,136,158,176, 207,221,253,383, ,12,15-Octadecatrienoic acid, 2,3- dihydroxypropyl ester, (Z,Z,Z) C27H44O ,67,79,95,109,155,173,2 32,261,291,321,352 Several active chemical constituents of C. colocynthis plant were recorded. They are grouped as alkaloids, flavonoids, saponins, tannins, carbohydrates, glycosides and essential oils. Plant based natural constituents can be derived from any part of the plant like stems, leaves, flowers, roots, fruits and seeds (Gordon and David, 2001). A number of plant secondary metabolites, including flavonoids and curcubitacins have previously been reported from C. colocynthis (Seger et al., 2005). It has been used in herbal treatment of diabetes (Karim et al., 2011), edema, bacterial infection and cancer. The aqueous pulp extract of the fruit is used for kidney, liver functions treatment (Rahbar and Nabipour, 2010). Conclusion From the results obtained in this study, it could be
9 Idan et al Table 1. Contd. 24 Triazido-(1,2,3,4,5-pentamethylcyclopenta-2,4- dienyl)-german C10H15GeN ,91,105,119,134,148,17 7,207,235,267,335 Anti-inflammatory 25 Ethyl iso-allocholate C26H44O ,69,81,95,213,253,400,4 18 Anti microbial agents and anti-inflammatory activity. 26 α-n-normethadol C20H27NO ,77,91,115,134,165,178, 208,239,277,297 Antioxidant activity. 27 Octadecanoic acid, 2-hydroxy-1- (hydroxymethyl)ethyl ester C21H42O ,73,98,134,185,267,284, 327,358 Antimicrobial and antiinflammatory.
10 3140 Afr. J. Biotechnol. Table 1. Contd. 28 Phthalic acid, decyl oct-3-yl ester C26H24O ,104,149,167,193,251,3 07 Anti-hyper cholesterol 29 1,2-Benzenedicarboxylic acid, bis(8- methylnonyl)ester C28H46O ,99,149,167,193,228,28 9,307,321,361,389,417 Preventing many diseases such as anti- (hypertension, cholesterol) 30 Phthalic acid, di(6-ethyl-3-octyl)ester C28H46O ,84,111,149,167,307 Biological activities like antimicrobial, antifungal and antioxidant activity 31 y-tocopherol C28H48O ,107,135,151,191,205,2 32,260,288,316,344,372 Antioxidant activity 32 1,4-Ethanonaphthalene dione,1,4a,5,8a-tetrahydro-4,5,7,10-6,9(4h) C20H28O2S ,77,91,105,135,149,187, 255,289, Vitamin E C29H50O ,69,91,121,165,205,246, 274,302,330,358,386,430 Antioxidant activity
11 Idan et al Figure 1. GC-MS profile of Citrullus colocynthis. Figure 2. Structure of methyl 6-oxoheptanoate present in the Citrullus colocynthis by using GC-MS analysis
12 3140 Afr. J. Biotechnol. Figure 3. Structure of hexanoic acid, 2-isopropyl-2-methyl-5-oxo-, methyl ester present in the Citrullus colocynthis by using GC-MS analysis. Figure 4. Structure of dodecanoic acid, 3-hydroxy present in the Citrullus colocynthis by using GC-MS analysis.
13 Idan et al Figure 5. Structure of benzofuran,2,3-dihydro present in the Citrullus colocynthis by using GC-MS analysis. Figure 6. Structure of 1,1-cyclopropanedimethanol, 2-methyl-α-phenyl present in the Citrullus colocynthis by using GC-MS analysis.
14 3144 Afr. J. Biotechnol. Figure 7. Structure of 12,15-octadecadiynoic acid, methyl ester present in the Citrullus colocynthis by using GC-MS analysis. Figure 8. Structure of (5ß)pregnane-3,20ß-diol, 14α,18α-[4-methyl-3-oxo-(1-oxa-4- azabutan present in the Citrullus colocynthis by using GC-MS analysis.
15 Idan et al Figure 9. Structure of 3-(N,N-dimethyllaurylammonio)propanesulfonate present in the Citrullus colocynthis by using GC-MS analysis. Figure 10. Structure of 2H-1-benzopyran-3,4-diol,2-(3,4-dimethoxyphenyl)- 3,4dihydro-6-met present in the Citrullus colocynthis by using GC-MS analysis.
16 3146 Afr. J. Biotechnol. Figure 11. Structure of 11,13-dihydroxy-tetradec-5-ynoic acid, methyl ester present in the Citrullus colocynthis by using GC-MS analysis. Figure 12. Structure of cyclopenta[1,3]cyclopropa[1,2]cycloheptan-3(3ah)- one,1,2,3b,6,7 present in the Citrullus colocynthis by using GC-MS analysis.
17 Idan et al Figure 13. Structure of 4-(2,4,4-Trimethyl-cyclohexa-1,5-dienyl)-but-3-en-2-one present in the Citrullus colocynthis by using GC-MS analysis. Figure 14. Structure of 1-tetradecanamine, N,N-dimethyl present in the Citrullus colocynthis by using GC-MS analysis.
18 3148 Afr. J. Biotechnol. Figure 15. Structure of α-d-glucopyranoside, O-α-Dglucopyranosyl-(1,fwdarw.3)-ß-D-fructo present in the Citrullus colocynthis by using GC-MS analysis. Figure 16. Structure of acetamide,n-methyl-n-[4-(3- hydroxypyrrolidinyl)-2-butynyl] present in the Citrullus colocynthis by using GC-MS analysis.
19 Idan et al Figure 17. Structure of 9-octadecenamide,(z) present in the Citrullus colocynthis by using GC-MS analysis. Figure 18. Structure of butyrophenone,2,3,4,6 -tetramethyl present in the Citrullus colocynthis by using GC-MS analysis
20 3150 Afr. J. Biotechnol. Figure 19. Structure of ethyl 5,8,11,14,-eicosatetraenoate present in the Citrullus colocynthis by using GC-MS analysis. Figure 20. Structure of 1H-cyclopropa[3,4]benz[1,2-e]ezulene -5,7b,9,9a tetrol,1a,1b,4,4a present in the Citrullus colocynthis by using GC-MS analysis.
21 Idan et al Figure 21. Structure of 9,12,15-octadecatrienoic acid present in the Citrullus colocynthis by using GC-MS analysis. Figure 22. Structure of 9,10-secocholesta -5,7,10(19)-triene-3,24,25,-triol,(3ß,5Z,7E) present in the Citrullus colocynthis by using GC-MS analysis.
22 3152 Afr. J. Biotechnol. Figure 23. Structure of 9,12,15-octadecatrienoic acid,2,3-dihydroxypropyl ester, (Z,Z,Z) present in the Citrullus colocynthis by using GC-MS analysis. Figure 24. Structure of triazido-(1,2,3,4,5-pentamethylcyclopenta-2,4-dienyl)- german present in the Citrullus colocynthis by using GC-MS analysis.
23 Idan et al Figure 25. Structure of ethyl iso-allocholate present in the Citrullus colocynthis by using GC-MS analysis. Figure 26. Structure of α-n-normethadol present in the Citrullus colocynthis by using GC-MS analysis.
24 3154 Afr. J. Biotechnol. Figure 27. Structure of octadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester present in the Citrullus colocynthis by using GC-MS analysis. Figure 28. Structure of phthalic acid, decyl oct-3-yl ester present in the Citrullus colocynthis by using GC-MS analysis.
25 Idan et al Figure 29. Structure of 1,2-benzenedicarboxylic acid, bis(8-methylnonyl)ester present in the Citrullus colocynthis by using GC-MS analysis. Figure 30. Structure of phthalic acid, di(6-ethyl-3-octyl)ester present in the Citrullus colocynthis by using GC-MS analysis.
26 3146 Afr. J. Biotechnol. Figure 31. Structure of γ-tocopherol present in the Citrullus colocynthis by using GC-MS analysis. Figure 32. Structure of 1,4-ethanonaphthalene -6,9(4H)-dione,1,4a,5,8atetrahydro-4,5,7,10 present in the Citrullus colocynthis by using GC-MS analysis.
27 Idan et al Figure 33. Structure of vitamin E present in the Citrullus colocynthis by using GC-MS analysis. Figure 34. Structure of octadecanoic acid present in the Citrullus colocynthis by using GC-MS analysis.
28 3148 Afr. J. Biotechnol. Table 2. Zone of inhibition (mm) of test bacterial strains to Citrullus colocynthis bioactive compounds and standard antibiotics. Bacteria Plant (Citrullus colocynthis)/antibiotics Citrullus colocynthis Streptomycin Rifambin Cefotoxime Klebsiella pneumonia 3.95± ± ± ±0.200 Proteus mirabilis 2.06± ± ± ±0.350 Pseudomonas eurogenosa 4.91± ± ± ±0.130 Staphylococcus aureus 2.95± ± ± ±0.101 Escherichia coli 3.00± ± ± ±0.240 concluded that C. 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. Conflict of interests The author(s) did not declare any conflict of interest. ACKNOWLEDGEMENT Special thanks to Prof. Abdul-Kareem, Babylon University, Faculty of Science for Women, for his special care. REFERENCES Al-Tameme HJ, Hameed IH, Idan SA, Hadi MY (2015a). 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SUMMARY The isolation and characterisation of four flavonoid constituents from the roots of Dalbergia congesta, not hitherto phytochemically examined, are presented in Chapter I. The petroleum ether extract,
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APPENDIX Reagents Alsever s solution Citric acid 0.55g Sodium citrate 8.0g D-glucose 20.5g Sodium Chloride 4.2g 1000 ml Barritt s reagent A α-naphthol 5.0g ethanol 95.0 ml α-naphthol was dissolved in ethanol
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CHAPTER 7 GENERAL DISCUSSION AND CONCLUSION 7.1 Introduction.. 127 7.2 Bioassay guided fractionation of the ethanol crude extract and isolated compounds..... 127 7.3 Biological evaluation of the isolated
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