WSN 49(2) (2016) 381-404 EISSN 2392-2192

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Gas chromatography mass spectrum and Fourier transform - infrared spectroscopy analysis of methanolic extract of Cressa cretica L. leaves

Aseel Muhammed Omran, Nidaa Adnan Abu-seraj, Ibtihal Muiz Al Husaini

Department of Biology, Babylon University, Hilla, Iraq E-mail address: [email protected]

ABSTRACT

Aims of this study were to investigation the presence of phytochemical compounds in the methanolic extract of Cressa cretica L. leaves by using GC-MS method and report the functional groups by using FT-IR spectroscopy. The identification of phytochemical compounds based on the peak area, retention time, molecular weight, molecular formula, MS fragment ions. Thirty four phytochemical compounds were identification in the methanolic extract of Cressa cretica leaves. The GC-MS analysis provide the existence of 5-Methyl-6-phenyltetrahydro-1,3-oxazine-2-thione, Lactose, 3-Deoxy-L-ribose-2,5-dibenzoate, Sarreroside, Pterin-6-carboxylic acid, Octadecadiynoic acid, methyl ester, d-Mannose, Dodecanoic acid, 3-hydroxy-, Geranyl isovalerate, Tetradecanoic acid , 6-epi- shyobunol, Paromomycin, Cis-9-Hexadecenoic acid, and others. The FT-IR analysis revealed the presence of Alkenes, aliphatic amines, nitro compounds, alkanes. These are chemical compounds are may be useful for various herbal formulation as antifungal, antibacterial, anti- inflammatory, antioxidant and others.

Keywords: FT-IR; GC-MS analysis; Leaves; Methanol; Cressa cretica

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Worl d Sci ent ific N ew s 49(2) (2016) 381-404

1. INTRODUCTION

Herbal medicines had usually used for treatment of diseases and for helth mentinance [1], plant based drugs have been used since immemorial time, which interesting with herbal products lead to grouth of medicinal plant industries [2]. Cressa cretica L. (convolvulaceae) is a small, dwarf shrub [3], roots are horizontal, geminate with lateral branches leading upward to produce aboveground parts. The leaf blade is 1-2 mm long, lanculate, ovate or elliptic to scale like. Flowers are solitary, white or pink axillary, 5-8 mm long [4]. Seeds are 3-4 mm long, glabrous, smooth and shining to reticulate, with dark, brown color [5].

Cressa cretica showed variation in productivity in response to environmental factors.

Net productivity and aboveground biomass were higher during the brief winter in comparison to summer [6]. C. cretica used in all parts as a paste and decoction to treated fungus infection, asthma, blood purifier and eczema [7]. Aqueous and alcoholic extracts of leaves of this plant have a very good activity against some microbial pathogens such as gram – positive, gram negative bacteria and some fungi species, such as Candida albicans, Aspergillus niger, and Penicillium chrysogenum [8]. The plant can be used as anti-tubercular, expectorant [9].

Ethanolic extract of C. cretica significantly reduced blood glucose, serum cholesterol in rats [4]. This plant also used to synthesis silver nanoparticles from silver nitrate using C. cretica leaf extract [10].

In the last few years gas chromatography – mass spectrometry has become firmly established as a key technology plat form for phytochemical profiling in plant [11-13]. Gas chromatography provides a very adequate technique for the separation of complex samples because this technique give a combination of speed, sensitivity and a high resolving power [14]. Fourier transform infrared spectrometry is a physico-chemical analytical technique and one of the most widely used methods to identify the structure of unknown composition or its functional group, and the intensity of the absorption spectra associated with molecular composition or content of the chemical group [15]. The present study involves an assessment using GC-MS and FT-IR spectroscopic techniques to investigate and determine the bioactive compounds in the leaves of C. cretica.

2. MATERIALS AND METHODS

Collection and preparation of plant material

The leaves were purchased from the gardens of Babylone University, Hilla city, after had cleaned and removal foreign materials, the leaves were washed twice with running tap water and once with distilled water and dried under shade for ten days at room temperature.

Dried leaves stored in airtight container to avoid the effect of humidity and then stored at room temperature until further use.

Preparation of sample about 20 gm of the plant sample powdered were soaked in 100 ml methanol for 16 h in a rotatory shaker. What man No.1 filter paper was used to separate the extract of plant. The supernatant were used for further phytochemical analysis [16]. It was again filtered through sodium sulphate in order to remove the traces of moisture.

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Gas chromatography mass spectrum analysis

The GC-MS analysis of the plant extract was made in a (QP 2010 Plus SHIMADZU) instrument under computer control at 70 eV [17-19]. About 1 μl of the methanol extract was injected into the GC-MS using a micro syringe , then the scanning was done for 45 min. As the compounds were separated, and removed from the column and entered a detector which was capable of creating an electronic signal whenever a compound was detected. The greater concentration in the sample, bigger was the signal obtained which then processed by the 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. 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 removed 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. The M/Z (Mass/Charge) ratio obtained was calibrated from the graph obtained, which was called the Mass spectrum graph which is the fingerprint of a molecule. Before analyzing the extract using gas chromatography and mass spectroscopy , the temperature of the oven and the flow rate of the gas used and 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 1ml per minute. The electron gun of mass detector liberated electrons having energy of about 70eV. The column employed here for the separation 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 [19,20].

Fourier transform infrared spectrophotometer (FTIR)

The powdered sample of the plant specimen was treated for FTIR spectroscopy (Shimadzu, IR Affinity 1, Japan). The sample was run at infrared region between 400 and 4000 nm [21,22].

3. RESULTS AND DISCUSSION

Gas Chromatography and Mass spectroscopy analysis of compounds was carried out in methanolic extract of C. cretica leaves, shown in Table 1.and the components corresponding to the peaks were determined as follows: 5-Methyl-6-phenyltetrahydro-1,3-oxazine-2-thione;

Eicosanoic acid, phenylmethyl ester; Lactose ; 3-Deoxy-L-ribose-2,5-dibenzoate; Adenosine , 4'-methylaminoformyl-4'-deshydroxymethyl-N-[3-is; Benzenemethanol, 4-hydroxy-α-[1- (methylamino) ethyl]; Benzenemethanol, 2-(2-aminopropoxy)-3-methyl-; Sarreroside; Pterin- 6-carboxylic acid; 12,15-Octadecadiynoic acid , methyl ester; d-Mannose; Dodecanoic acid, 3-hydroxy-; Cyclopenta[1,3] cyclopropa[1,2]cyclohepten-3(3Ah)-one, 1,2; Desulphosinigrin;

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Geranyl isovalerate; Cyclopentanemethylamine, 2-isopropylidene-N,N,5-trimethyl-;

Tetradecanoic acid; Ppropiolic acid, 3-(1-hydroxy-2-isopropyl-5-methylcyclohexyl);

Acetamide, N-methyl-N-[4-(3-hydroxypyrrolidinyl)-2butynyl]; 6-epi-shyobunol; 2,7- Diphenyl-1,6-dioxopyridazino[4,5:2',3']pyrrolo[4',5'-d]pyrid; 3,7,11,15-Tetramethyl-2- hexadecan-1-ol; Paromomycin; Cis-9-Hexadecenoic acid; γ-Linolenic acid , methyl ester;

Estra-1,3,5(10)-trien-17β-ol; Ethyl iso-allocholate; 7,10-Octadecadienoic acid, methyl ester;

Cholestan-3-ol,2-methylene-,(3β,5α)-; Octadecanoic acid; Strychane, 1-acetyl-20α-hydroxy- 16-methylene-; 8,8'-Trimethoxy -3 -piperidyl-2,2'-binaphthalene-1,1',4,4'-tetra; Spirost-8-en- 11-one,3-hydroxy-,(3β,5α,14β,20β,22β,25R)-; Campesterol (Figer 1-34).

Fourier- transform infrared analysis of methanolic extract of cressa cretica leaves proved the presence of Alkenes, aliphatic amines, nitro compounds, amines, alkanes, (Table 2; Figer 35). Among the identified phytocompounds have the property of antioxidant and antimicrobial activities [23,24].

Plant based antimicrobials have enormous therapeutic potential as they can serve the purpose with lesser side effects. Continued further exploration of plant derived antimicrobials is needed today. [25] and [5] investigate the activity of alcoholic extract of cressa cretica as anti fungul. Chaudhary reported that the ethanolic extract of C. cretica was significantly reduced blood glucose and this extract have a high anti diabetic potential.

Figure 1. 5Methyl-6phenyltetrahydro1,3oxazine Figure 2. Eicosanoic acid , phenylmethyl -2-thione in the leaf extract of C. cretica L. ester in the leaf extract of C. cretica L.

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Figure 3. Lactose in the leaf extract Figure 4. 3-Deoxy-L-ribose-2,5-dibenzoate in of C. cretica L. the leaf extract of C. cretica L.

Figure 5. Adenosine , 4'-methylaminoformy Figure 6. Benzenemethanol,4-hydroxy-α- l-4'-deshydroxymethyl-N-[3-is in the leaf -[1-(methylamino)ethyl]-,(R* in the leaf extract of C. cretica L. extract of Cressa cretica L

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Figure 7. Benzenemethanol , 2-(2-aminopropoxy) Figure 8. Sarreroside in the leaf 3-methyl- in the leaf extract of Cressa cretica L. extract of Cressa cretica L.

Figure 9. Pterin-6-carboxylic acid in Figure 10. 12,15-Octadecadiynoic acid, methyl the leaf extract of C. cretica L. ester in the leaf extract of C. cretica L.

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Figure 11. d-Mannose in the leaf extract Figure 12. Dodecanoic acid , 3-hydroxy of C. cretica L. in the leaf extract of C.a cretic

Figure 13. Cyclopenta[1,3]cyclopropa[1,2 Figure 14. Desulphosinigrin in the cyclohepten-3(3Ah)-one,1,2 in the leaf leaf extract of C. cretica L.

extract of C. cretica L.

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Figure 15. Geranyl isovalerat Figure 16. Cyclopentanemethylamine.

in the leaf of C. cretica L. 2-isopropylidene- N,N,5-trimethyl-, in the leaf of C.cretica L

Figure 17. Tetradecanoic acid in the leaf Figure 18. Ppropiolic acid ,3-(1-hydroxy-2- extract of C. cretica L. isopropyl 5-methylcyclohexyl)- in the leaf extract of C. cretica L.

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Figure 19. Acetamide , N-methyl-N-[4- Figure 20. 6-epi-shyobunol in the (3-hydroxypyrrolidinyl)-2-butynyl]- in leaf extract of C. cretica L.

the leaf extract of C. critica L.

Figure 21. 2,7-Diphenyl-1,6-dioxopyridazino Figure 22. 3,7,11,15Tetramethyl-2hexadecan- [4,5:2',3']pyrrolo[4',5'-d]pyrid in the leaf 1-ol in the leaf extract of C. cretica L.

extract of C. cretica L.

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Figure 23. Paromomycin in the leaf Figure 24. Cis-9-Hexadecenoic acid extract of C. cretica L. in the leaf extract of C. cretica L.

Figure 25. γ-Linolenic acid , methyl ester in Figure 26. Estra-1,3,5(10)-trien-17β-ol the leaf extract of C. cretica L. in the leaf extract of C. cretica L.

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Figure 27. Ethyl iso-allocholate in the leaf Figure 28. 7,10-Octadecadienoic acid, methyl extract of C. cretica L. ester in the leaf extract of C. cretica L.

Figure 29. Cholestan-3-ol,2-methylene-,(3β,5α)- Figure 30. Octadecanoic acid in the in the leaf extract of C.cretica L. leaf extract of C. cretica L.

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Figure 31. Strychane, 1-acetyl-20α-hydroxy-16 Figure 32. 3',8,8'-Trimethoxy-3-piperidyl-2 -methylene- in the leaf extract of C. cretica L. 2'-binaphthalene-1,1',4,4'-tetra, in the leaf extract of C. cretica L.

Figure 33. Spirost-8-en-11-one,3-hydroxy-, Figure 34. Campesterol in the leaf (3β,5α,14β,20β,22β,25R)- in the leaf extract extract of C. cretica L.

of C. cretica L.

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Table 1. Major phytochemical compounds identified in methanolic extract of Cressa ctretica Linn Leaves.

Pharmacolog-ical actions

MS Fragment- ions

Chemical structure

Exact Mass

Molecular Weight

RT (min)

Phytochemical compound

Serial No. 57,77,91,97, 117,132,147, 174,207

207.071785

2073.367

5-Methyl-6- phenyltetrahydro-1,3- oxazine-2-thione

1. No active report

57,71,91,108,126,1 47,167,207,281

402.349781

4023.968

Eicosanoic acid , phenylmethyl ester

2. Preservative [26]

60,73,85,91,97,126,145,16 3,191

342.11621

3424.735

Lactose

3. No active report

51,77,92,105,122,13 6,165,207

342.110338

3424.849

3-Deoxy-L-ribose- 2,5-dibenzoate

4.

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No active report

58,76,85,97,120,148,16 3,206,240,282

441.121923

4416.274

Adenosine , 4'- methylaminoformyl-4'- deshydroxymethyl-N- [3-is

5. No active report

58,65,77,95,121,147,181

181.110279

1816.903

Benzenemethanol,4-hydroxy- α-[1-(methylamino)ethyl]- ,(R*

6. Anti-nociceptive effect. [21]

58,65,77,91,105,121,135,1 52,178,195

195.125929

1958.168

Benzenemethanol , 2-(2- aminopropoxy)-3-methyl-

7. No active report

57,74,113,191,229,271,35 4,401,445

562.277798

5628.631

Sarreroside

8. Anti-psychotic, mood- stabilizer and anti- parasite [21]

57,69,93,105,122,149,1 63,177,207

207.039239

2079.072

Pterin-6-carboxylic acid

9.

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No active report

55,67,74,79,91,119 ,133,147,161,178,2 05,220

290.22458

2909.564

12,15- Octadecadiynoic acid , methyl ester

10. Anti-allergic and anti-bacterial [22]

60,73,103,149

180.063388

18010.714

d-Mannose

11. No active report

55,69,83,96,112,12 3,138,151,180,200

216.1725445

21610.960

Dodecanoic acid , 3-hydroxy-

12 Anti - pain effect. [21]

55,69,78,91,105,11 9,133,147,162,190

190.135765

19011.538

Cyclopenta[1,3]cyc lopropa[1,2]cycloh epten-3(3Ah)-one ,1,2

13 anticancer activity [27]

60,73,85,103,127,145,163,2 13,262

279.077658

27911.567

Desulphosinigrin

14 antifungal activity [28]

57,69,85,93,103,12 1,129,136,154,168, 183,198,219

238.19328

23812.253

Geranyl isovalerate

15

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No active report

58,79,107,136,164

181.18305

18112.580

Cyclopentanemethylamine , 2-isopropylidene-N,N,5- trimethyl-,

16 Antioxidant, cancer preventive,nematicide,hy pocholesterolemic, lubricant [27 ]

55,60,73,83,97,115,129, 143,157,171,185,199,21 1,228

228.20893

22812.911

Tetradecanoic acid

17 No active report

55,69,81,95,109,121,13 5,150,163,178,191,206

224.141245

22413.209

Ppropiolic acid , 3-(1- hydroxy-2-isopropyl-5- methylcyclohexyl)-

18 No active report

56,68,124,137,167,192

210.136827

21013.432

Acetamide , N-methyl- N-[4-(3- hydroxypyrrolidinyl)-2- butynyl]-

19 No active report

55,67,81,93,109,121 ,136,161,207,222

222.198365

22213.787

6-epi-shyobunol

20

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Anti- angiogenic effect and anti- tumor efficacy [22]

51,65,77,93,105,119,149,165,187,21 1,224,238,267,281,327,355

355.106924

35514.170

2,7-Diphenyl-1,6- dioxopyridazino[4,5:2',3']pyrrolo[4', 5'-d]pyrid

21 Antimicrobial anti- inflammatory [29]

55,71,81,95,109,123, 137,151,179,278

296.307917

29614.113

3,7,11,15- Tetramethyl-2- hexadecan-1-ol

22 Anti-bacterial Agents. [22]

57,67,80,94,109,124,145, 227,252,277,303

615.296303

61514.359

Paromomycin

23 Flavoring agent , Anti- oxidant . [24]

55,69,83,97,111,123, 137,192,236,254

254.22458

25414.502

Cis-9-Hexadecenoic acid

24 Antihistaminic, Anticoronary, Insectifuge ,Antieczemic, [29]

55,67,79,93,107,12 1,135,150,163,175, 194,207,235,260

292.24023

29214.748

γ-Linolenic acid , methyl ester

25

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Anti- arrhythmic activities [22]

57,73,85,97,107,129, 157,185,213,241,256

256.182714

25614.994

Estra-1,3,5(10)-trien- 17β-ol

26 Antimicrobial Diuretic Anti- inflammatory Antiasthma [30;31]

55,69,81,95,253,367,400,418

436.318874

43615.704

Ethyl iso-allocholate

27 No active report

55,67,81,95,109,121,1 50,164,205,220,234,26 3,294

294.25588

29416.173

7,10-Octadecadienoic acid , methyl ester

28 No active report

69,81,95,105,121,133,16 1,175,203,227

400.370516

40016.310

Cholestan-3-ol,2- methylene-,(3β,5α)-

29 Cancer preventive Insectifuge [32]

60,73,83,97,115,129,143,157, 171,185,199,227,241,255,284

284.27153

28416.802

Octadecanoic acid

30

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No active report

57,70,88,130,166,239,281,338

338.199429

33819.543

Strychane , 1-acetyl-20α-hydroxy-16- methylene-

31 Anticancer, antiarthritic and anti- inflammatory [22]

57,71,112,149,167,223,279,313,328

487.163101

48720.012

3',8,8'-Trimethoxy-3-piperidyl-2,2'- binaphthalene-1,1',4,4'-tetra

32 Estrogenic, progesterogenic and anti- inflammatory effects [21]

57,69,95,135,187,207,229, 281,299,314,356,395,428

428.29266

42819.761

Spirost-8-en-11-one,3- hydroxy- ,(3β,5α,14β,20β,22β,25R)

33 No active report

55,81,145,161,213,255, 289,315,382,400

400.370516

40027.359

Campesterol

34

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Table 2. FT-IR peak values of solid analysis of methanolic extract of Cressa cretica l.

Group frequency

Functional group assignment

Type of Vibration

Bond

Type of Intensity

Corr. Area

AreaBase (L)

Base (H)

Corr. Intensity

Intensity

Peak (Wave Number cm-ˡ) No.

650-1000

Alkenes

Bending

=C–H

Strong

0.258

5.535

648.68

686.66

2.513

70.544

667.37

1.

650-1000

Alkenes

Bending

=C–H

Strong

0.196

5.577

705.95

750.31

2.156

72.699

719.45

2.

650-1000

Alkenes

Bending

=C–H

Strong

0.102

1.967

860.25

885.33

1.879

82.145

875.68

3.

1020-1250

aliphatic amines

Stretch

C–N

Medium

8.040

26.632

927.76

083.99

15.580

58.525

1026.13

4.

1020-1250

aliphatic amines

Stretch

C–N

Medium

0.429

7.559

1085.92

139.93

2.504

68.167

1095.57

5.

1020-1250

aliphatic amines

Stretch

C–N

Medium

0.243

4.258

1141.86

192.01

1.333

78.651

1147.65

6.

1020-1250

aliphatic amines

Stretch

C–N

Medium

0.168

2.544

1213.231

253.73

1.210

85.359

1244.95

7.

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4. CONCLUSION

Cressa cretica Linn. is a native plant of Iraq. Thus the GC-MS analysis of methanolic extract of leaves of this plant showed a highly complex profile containing approximately thirty four components. It contain phytochemicals which may be useful for various herbal formulation as anti-inflamatory, anti-bacterial, anti-fungal and others.

References

[1] Sahito, S.R.;Memon, M.A., Kazi, T.G. and Gazi, G.H. Evaluation of mineral Contents in medicinal plant Azadirachta indica (neem). J. Chem. Soc. Pak. 25(2): 139-143, 2003.

[2] Barnes, J., Anderson, L.A. and Philipson, J.D. Herbal Medicines. A guide for Healthcare Professionals, thirded. Pharmaceutical Press, London, 2007.

[3] Rani, S., Chaudhary, S., Singh, P., Mishra, G., Jha, K.K., and Khosa, R.L. Cressa cretica Linn: An Important Medecinal Plant- A Review on Its Traditional Uses, Phytochemical and Pharmacological Properties. J. Nat. Prod. Plant Resour., 1(1): 91- 100, 2011.

[4] Chaudhary, S., Khosa, R.L., Jha, K.K. and Verma, N. Evaluation of Activity of Cressa cretica Linn in Alloxan Induced Diabetes in Rats. Pharmacology online 3: 181-188, 2010.

1290-1360

nitro compounds

Symmetric stretch

N–O

Medium

0.203

2.394

1294.24

1332.81

2.753

84.823

1317.83

8.

1580 -1650

amines

Bending

N–H

Medium

0.022

2.394

1581.63

1618.28

1.193

82.524

1616.35

9.

2850-3000

alkanes

Stretch

C-H

Medium

0.733

2.350

2785.21

2873.94

11.744

82.796

2848.86

10.

2850-3000

alkanes

Stretch

C-H

Medium

2.702

5.397

2875.86

2983.88

17.023

77.067

2918.30

11.