Using Gas Chromatography-Mass Spectrometry (GC-MS) Technique for Analysis of Bioactive Compounds of Methanolic Leaves extract of Lepidium sativum

 

Hussein J. Hussein¹, Imad Hadi Hameed*², Mohammed Yahya Hadi ³

¹Department of Biology, College of Science for women, University of Babylon, Iraq

²College of Nursing, University of Babylon, Iraq

³College of Biotechnology, Al-Qasim Green University, Iraq

 

ABSTRACT:

Cress (Lepidium sativum), sometimes referred to as garden cress to distinguish it from similar plants also referred to as cress (from old Germanic cresso which means sharp, spicy), is a rather fast-growing, edible herb. The objective of this study was analysis of the secondary metabolite products. Bioactives are chemical compounds often referred to as secondary metabolites. Ninteeth bioactive compounds were identified in the methanolic extract of Lepidium sativum. The identification of bioactive chemical compounds is based on the peak area, retention time molecular weight and molecular formula. GC-MS analysis of Lepidium sativum revealed the existence of the Glycerin, Monoethanolamine, 1-Deoxy-d-mannitol, 1-Nitro-2-propanol , 2-Butanamine , (S)-,  Furfural, Allyl isothiocyanate , Paromomycin, 2-Hydroxy-2-(5-methylfuran-2-yl)1-phenylethanone , 3,6-Diazahomoadamantan-9-one Hydrazone , 2,3,4-Trimethoxycinnamic acid , 2-Naphthalenol , 2,3,4,4a,5,6,7-octahydro-1,4a-dimethyl-7-(2)- , cis-Vaccenic acid , 9-Octadecenamide , γ-Tocopherol , Phthalic acid , decyl oct-3-yl ester , Ergosta-5,22-dien-3-ol,acetate , ( 3β,22E)- , Campesterol and Cholest-5-en-3-ol ,24-propylidene-,(3β). GC-MS is widely used in pharmaceutical industries for analytical research and development, quality control, quality assurance, production, pilot plants departments for active pharmaceutical ingredients (API), bulk drugs and formulations.

 

 

 

INTRODUCTION:

Lepidium sativum have been widely used to treat a number of diseases in traditional system of medicine throughout Iraq. It is useful in case of lumbago or any other pains about the loins through rheumatism ^1-8. Its external application with lime juice for relief of internal inflammation and rheumatic pain is useful. Garden cress is genetically related to watercress and mustard, sharing their peppery, tangy flavor and aroma. In some regions, garden cress is known as mustard and cress, garden pepper cress, pepperwort, pepper grass, or poor man's pepper.

This annual plant can reach a height of 60 cm (~24 inches), with many branches on the upper part. The white to pinkish flowers are only 2 mm (1/12 of an inch) across, clustered in branched racemes. It is useful in asthma, cough, diarrhoea, dysentry, skin disease, blood disorder ^9-20. It is having hot and bitter property. It is consider as tonic, galactagogue, aphrodisiac, destroys vata and kapha, cures dysentery, good for pain in abdomen, blood and skin disease and tumours. The fresh fruit is good for injury, skin and eye disease. Its seeds are hot, and leaves hot and dry, diuretic, aphrodisiac, good in inflammation and spleen disease, in chest complaints, bronchitis, and rheumatism and muscular pains. The root is used in secondary syphilis and tenesmus. According to Bellow the seeds are also considered to be galactagogue in the Punjab, and are administered after being boiled with milk, to cause abortion. It is used in the treatment of asthma, cough and bleeding piles, leaves are mildly stimulant and diuretic and useful in scorbutic diseases and liver complaints ^21-38. The root is used in secondary syphilis and tenesmus. The seeds of the plant are rubefacient, galactogogue, emmenagogue, laxative, tonic, aphrodisiac and diuretic. They are used in poultice for hurts and sprains. The aim of this study was analysis of the secondary metabolite products of Lepidium sativum.

 

MATERIALS AND METHODS:

Gas chromatography – Mass Spectrum analysis

Interpretation of mass spectrum was conducted using the database of National Institute of Standards and Technology (NIST, USA). The database consists of more than 62,000 patterns of known compounds. The spectrum of the extract was matched with the spectrum of the known components stored in the NIST library. Lepidium sativum GC–MS analysis were carried out in a GC system (Agilent 7890A series, USA). The flow rate of the carrier gas, helium (He) was set to beat 1 mL min−1, split ratio was 1:50. The injector temperature was adjusted at 250◦C, while the detector temperature was fixed to280◦C. The column temperature was kept at 40◦C for 1 min followed by linear programming to raise the temperature from 40◦to 120◦C (at 4 Cº min−1with 2 min hold time), 120 Cº to 170 Cº (at 6 Cº min−1with 1 min hold time) and 170 Cº to 200 Cº (at10◦C min−1with 1 min hold time) ^39-52. The transfer line was heated at 280 Cº. Two microliter of FAME sample was injected for analysis. Mass spectra were acquired in scan mode (70 eV); in the range of 50–550 m/z.

 

Statistical analysis

Results of the study were based on analysis of variance (ANOVA) using Statistica Software. A significance level of 0.05 was used for all statistical tests.

 

 

 

Table 1. Major phytochemical compounds identified in methanolic extract of Lepidium sativum.

Serial No.	Phytochemical compound	RT (min)	Molecular Weight	Exact Mass	Chemical structure	MS Fragment- ions	Pharmacological actions
1	Glycerin	3.31	92	92.047344		61,74	anti-inflammatory
2	Monoethanolamine	3.356	61	61.052764		61	Anti-Bacterial Agents
3	1-Deoxy-d-mannitol	3.453	166	166.084124		61,73,103,166	antispasmodic
4	1-Nitro-2-propanol	3.505	105	105.042593		61,69,90,104	antimicrobial
5	2-Butanamine , (S)-	3.59	73	73.0891495		58,72	New chemical compound
6	Furfural	3.676	96	96.021129		51,67,96	Anti inflammatory activity and analgesic activity
7	Allyl isothiocyanate	3.894	99	99.0142703		58,72,84,99	antimicrobial activity
8	Paromomycin	4.414	615	615.296303		57,67,80,94,109,191,227,259,292,324	Antimicrobial activity
9	2-Hydroxy-2-(5-methylfuran-2-yl)1-phenylethanone	4.334	216	216.078644		51,77,105,122,137,180,214	anticancer activity
10	3,6-Diazahomoadamantan-9-one Hydrazone	8.797	180	180.137497		58,72,95,121,180	Anti-microbial
11	2,3,4-Trimethoxycinnamic acid	13.964	238	238.084124		53,77,107,163,179,207,238	Anti-stress effects
12	2-Naphthalenol , 2,3,4,4a,5,6,7-octahydro-1,4a-dimethyl-7-(2)-	14.308	238	238.19328		55,67,81,107,123,161,177,205,220,238	anti- inflammatory
13	cis-Vaccenic acid	15.423	282	282.25588		55,69,83,97,111,123,165,193,222,264,282	anti-asthmatic, anti-inflammatory
14	9-Octadecenamide	17.346	281	281.271864		55,72,83,122,220,281	anti- inflammatory action
15	γ-Tocopherol	22.295	416	416.36543		57,69,107,151,191,246,288,330,372,416	antioxidant activity
16	Phthalic acid , decyl oct-3-yl ester	21.906	418	418.30831		57,104,149,167,193,251,307	anti-bacterial activity
17	Ergosta-5,22-dien-3-ol,acetate , ( 3β,22E)-	23.119	440	440.36543		55,67,91,145,213,255,281,327,380	New chemical compound
18	Campesterol	23.543	400	400.370516		55,81,145,213,255,289,315,400	anti-inflammatory, and in vitro cytotoxic activities
19	Cholest-5-en-3-ol ,24-propylidene-,(3β)-	24.195	426	426.386166		55,69,95,229,281,314,341,393,426	Antibacterial activity

 

 

 

 

Figure 1: GC-MS chromatogram of methanolic extract of Lepidium sativum.

 

 

Figure 2: Mass spectrum of  Glycerin   with Retention Time (RT)= 3.310

 

Figure 3: Mass spectrum of   Monoethanolamine  with Retention Time (RT)= 3.356

 

Figure 4: Mass spectrum of   1-Deoxy-d-mannitol  with Retention Time (RT)= 3.453

 

Figure 5: Mass spectrum of  1-Nitro-2-propanol   with Retention Time (RT)=3.505

 

Figure 6: Mass spectrum of  2-Butanamine , (S)-   with Retention Time (RT)= 3.590

 

 

Figure 7: Mass spectrum of  Furfural   with Retention Time (RT)= 3.676

 

 

Figure 8: Mass spectrum of  Allyl isothiocyanate   with Retention Time (RT)= 3.894

 

Figure 9: Mass spectrum of   Paromomycin  with Retention Time (RT)= 4.414

 

 

Figure 10: Mass spectrum of  2-Hydroxy-2-(5-methylfuran-2-yl)1-phenylethanone   with Retention Time (RT)= 4.334

 

 

Figure 11: Mass spectrum of  3,6-Diazahomoadamantan-9-one Hydrazone with Retention Time (RT)= 8.797

 

Figure 12: Mass spectrum of  2,3,4-Trimethoxycinnamic acid  with Retention Time (RT)= 13.964

 

Figure 13: Mass spectrum of  2-Naphthalenol , 2,3,4,4a,5,6,7-octahydro-1,4a-dimethyl-7-(2)-  with Retention Time (RT)= 14.308

 

Figure 14: Mass spectrum of cis-Vaccenic acid  with Retention Time (RT)=15.423

 

Figure 15: Mass spectrum of  9-Octadecenamide  with Retention Time (RT)=17.346

 

 

Figure 16: Mass spectrum of  γ-Tocopherol  with Retention Time (RT)= 22.295

 

Figure 17: Mass spectrum of  Phthalic acid , decyl oct-3-yl ester  with Retention Time (RT)=21.906

 

Figure 18: Mass spectrum of   Ergosta-5,22-dien-3-ol,acetate , ( 3β,22E)-  with Retention Time (RT)=23.119

 

 

Figure 19: Mass spectrum of   Campesterol  with Retention Time (RT)= 23.543

 

Figure 20: Mass spectrum of Cholest-5-en-3-ol ,24-propylidene-,(3β)-   with Retention Time (RT)=24.195

 

 

RESULTS AND DISCUSSION:

Identification of biochemical compounds

Analysis of compounds was carried out in methanolic extract of Lepidium sativum, shown in Table 1. The GC-MS chromatogram of the peaks of the compounds detected was shown in Figure 1. Chromatogram GC-MS analysis of the methanol extract of Lepidium sativum showed the presence of thirty one major peaks and the components corresponding to the peaks were determined as follows. All peaks were determined to be Glycerin , Monoethanolamine , 1-Deoxy-d-mannitol , 1-Nitro-2-propanol , 2-Butanamine , (S)- ,  Furfural , Allyl isothiocyanate , Paromomycin, 2-Hydroxy-2-(5-methylfuran-2-yl)1-phenylethanone, 3,6-Diazahomoadamantan-9-one Hydrazone, 2,3,4-Trimethoxycinnamic acid, 2-Naphthalenol, 2,3,4,4a,5,6,7-octahydro-1,4a-dimethyl-7-(2)-, cis-Vaccenic acid, 9-Octadecenamide , γ-Tocopherol , Phthalic acid , decyl oct-3-yl ester , Ergosta-5,22-dien-3-ol,acetate , ( 3β,22E)- , Campesterol and Cholest-5-en-3-ol ,24-propylidene-,(3β). Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase. Flowers are also much prized by some invalids being palatable and beautiful root is used in secondary syphilis and tenesmus. Seeds are diuretic, alterative, tonic, aphrodisiac, carminative, galaetogogue, and emmenogogue. Gas chromatography has a very wide field of applications. But, its first and main area of use is in the separation and analysis of multi component mixtures such as essential oils, hydrocarbons and solvents. Intrinsically, with the use of the flame ionization detector and the electron capture detector (which have very high sensitivities) gas chromatography can quantitatively determine materials present at very low concentrations. It follows, that the second most important application area is in pollution studies, forensic work and general trace analysis. Because of its simplicity, sensitivity, and effectiveness in separating components of mixtures, gas chromatography is one of the most important tools in chemistry. It is widely used for quantitative and qualitative analysis of mixtures, for the purification of compounds, and for the determination of such thermo chemical constants as heats of solution and vaporization, vapor pressure, and activity coefficients ^51-55. A knowledge of the chemical constituents of plants is desirable not only for the discovery of therapeutic agents, but also because such information may be of great value in disclosing new sources of economic phytocompounds for the synthesis of complex chemical substances and for discovering the actual significance of folkloric remedies. Higher plants as sources of bioactive compounds continue to play a dominant role in the maintenance of human health ^55-65.

 

CONCLUSION:

In recent years GC-MS studies have been increasingly applied for the analysis of medicinal plants as this technique has proved to be a valuable method for the analysis of non-polar components and volatile essential oil, fatty acids, lipids and alkaloids.

 

ACKNOWLEDGEMENT:

I thank Dr. Amean A. Al-yasiri, College of Nursing, for valuable suggestions and encouragement.

 

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Accepted on 28.09.2017           © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(11): 3981-3989.