International Journal of Advanced Life Sciences (IJALS)
ISSN 2277 – 758X
Krishnaveni and Saranya, IJALS, Volume (9) Issue (2) May - 2016. RESEARCH ARTICLE
Gas chromatography-mass spectrophotometry analysis of phytochemicals in Brassica hirta seeds M. Krishnaveni* and S. Saranya Department of Biochemistry, School of Bio-Sciences, Periyar University, Salem – 11, Tamilnadu, E-mail :
[email protected]
Corresponding Author M. Krishnaveni Department of Biochemistry, School of Bio-Sciences, Periyar University, Salem – 11, Tamilnadu Email : :
[email protected] Article History Received on 25 December, 2015; Received in revised form 16 January, 2016; Accepted 20 April, 2016
Abstract Phytochemicals are chemicals of the plant, synthesized naturally in bark, leaves, stem, root, flower, fruits, seeds, etc. Brassica sp. progressively used as a winter, rotational cover crops among the vegetable. Major producers of mustard seeds are Canada, Hungary, Great Britain, India, Pakistan, United States. Since, it is an economically important plant, care was given to utilize it completely as it offers many therapeutic applications. In addition to this, the validation and identification of phytoconstituents forms a major area of pharmaceutical science. Hence, the Brassica hirta seeds were assessed for its phytochemicals through gas chromatography –mass spectrophotometry analysis and its functional groups, compounds were analysed using Fourier transform infrared analysis. The gas chromatography analysis showed higher peaks for Erucic acid (19.85), n-Hexadecanoic acid (13.06), Ethanone, 1-(3,4-dimethoxy phenyl)-, 5-Hydroxymethyl furfural (11.98) having medicinal properties. The peak area percent was moderate for compounds like cis-13-Eicosenoic acid (7.34), 9 - Octa decenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl) ethyl ester (6.83), Campesterol (6.58), 9,12- Octa decadienoic acid (Z,Z)- (5.74), γTocopherol (3.58). The peak area percent was low for Iberin nitrile (1.97), 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester (1.10). The peak area percent was very low for 4-Hydroxy-3-methylacetophenone (0.58), cis-13Eicosenoic acid (0.02). The results of Fourier transform infrared analysis showed compounds like phenol, alcohol, alkene imparting health benefits to the society that found application in biomedicine. The present research showed that Brassica hirta seeds could be a good resource in the synthesis of novel drugs as it offers hypolipidemic, analgesic, anti-inflammatory, bactericidal, fungicidal and other activities. Keywords : Brassica hirta, fourier transform infrared, Gas chromatograph-mass spectrophotometry, phytochemicals and Seeds
Introduction Medicinal plants are great resource gaining attention in developing countries recently as it offers several benefits to humans towards the treatment of diseases. Most primitive indications to mustard in India began with the story of Buddha in 5th century BCE. The Brassica genus comprise the mustard group. The phrase “Brassica seed” represented to whole seeds of Brassica genus with reference to the family Brassicaceae / Cruciferae. The varieties of
Brassica include Brassica alba, Brassica hirta, Brassica juncea and Brassica nigra. The genus, species name obey with the International Code of Plant Nomenclature. White mustard called as Brassica hirta or Sinapis alba is an annual plant growing up to 70 cm high with stalkless pinnate leaves, (Webb, 1996) grown commercially for its seeds, fodder crop, manure, oil, biofuel, fumigant etc. The seeds are hard round about 1.0 to 1.5 mm in diameter (Balke et al., 2000) with a color varies from beige or yellow to
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International Journal of Advanced Life Sciences (IJALS)
ISSN 2277 – 758X
Krishnaveni and Saranya, IJALS, Volume (9) Issue (2) May - 2016. RESEARCH ARTICLE light brown. Mustard oil is used for body massage during excessive winter, as it keeps the body warm and moist. The plant is used in the treatment of skin disorder, anaemia (sickle cell), bone resorption, nasal dysfunction, angiogenesis, metastasis and several other disorders. Hence, it was decided to study the phytochemicals present in the Brassica hirta seeds through gas chromatography-mass spectrophotometry and also its functional group was analyzed using Fourier transform infrared analysis. Materials and methods Sample collection The Brassica hirta seed was purchased from a local shop at Salem. 25 gm of powdered seed was extracted with ethanol. The extracted sample was used for phytochemical analysis through Gas Chromatography-Mass Spectrophotometry. The seed purchased was identified for its botanical name through online sources. Analytical method Gas Chromatography - Mass Spectrophotometry Gas Chromatography-Mass Spectrophotometry was performed on a Scion 436-GC Bruker carrying Triple quadruple mass spectrophotometer with fused silica capillary column BR - 5MS (5% Diphenyl 95% Dimethyl poly siloxane), 30m x 0.25mm ID x 0.25m df. The column oven temperature program was as follows: 80°C hold for 2 min, Up to 160°C at the rate of 20°C/min-No hold, Up to 280°C at the rate of 5°C / min-No hold, Up to 300°C at the rate of 20°C/min-10 min hold, Injector temperature 280°C, Total GC running time was 36min. The inlet temperature was set at 280°C, source temperature 250°C; ionization mode, ionization at 70-eV ionization energy; For single scan analysis, the scan range was set from m/z 40 to 600; Solvent Delay: 0-3.5 min; and the injection volume was 2μl. The GC-MS/MS
was performed by the Institute of crop processing technology, Tanjavur. Fourier transform infrared analysis The Fourier transform infrared spectrum was recorded using Bruker Tensor 27 spectrometer in the wavelength range 400-4000 cm-1 by potassium bromide pellet technique with a resolution and scanning speed of 4 cm-1 and 2 mm / sec respectively. Results and Discussion The results of compounds identified are shown in Table - 1. Table.1 showed the results of phytochemicals present in the ethanol extract of Brassica hirta seeds. Totally 15 compounds were identified in Brassica hirta seeds. The molecular weight, molecular formulae peak area percent, retention time, were discussed as follows : The name of the compounds, molecular formulae, molecular weight identified through GC-MS/MS were given as follows: Compound 1: 5Hydroxy methyl furfural (C6H6O3, 126 ), Compound 2 : 4 - Hydroxy-3-methyl acetophenone (C9H10O2, 150), Compound 3: Iberin nitrile (C5H9NOS,131), Compound 4: Ethanone, 1-(3,4-dimethoxyphenyl)(C10H12O3, 180), Compound 5: 1,2-Benzene dicarboxylic acid, bis (2-methylpropyl) ester (C16H22O4, 278), Compound 6: n-Hexa decanoic acid (C16H32O2, 256) , Compound 7: 9,12-Octa decadienoic acid (Z,Z)(C18H32O2,280), Compound 8: cis-13-Eicosenoic acid (C20H38O2, 310), Compound 9: Tetracaine (C15H24N2O2, 264), Compound 10: Erucic acid (C22H42O2, 338), Compound 11: 9-Octa decenoic acid (Z)-, 2-hydroxy-1(hydroxymethyl) ethyl ester (C21H40O4, 356), Compound 12: γ-Tocopherol (C28H48O2,416) , Compound 13: cis-13-Eicosenoic acid (C20H38O2, 310), Compound 14: Stigmasterol (C29H48O,412), Compound 15: Campesterol (C28H48O, 400). Its peak area percent were high for Erucic acid (19.85), n-Hexadecanoic acid (13.06), Ethanone, 1-(3,4- dimethoxyphenyl)-
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ISSN 2277 – 758X
International Journal of Advanced Life Sciences (IJALS)
Krishnaveni and Saranya, IJALS, Volume (9) Issue (2) May - 2016. RESEARCH ARTICLE Table - 1. The results of compounds identified
S.No
RT
1 2 3 4 5
6.02 7.25 8.24 10.74 14.29
6 7 8 9 10 11
15.75 18.42 21.17 23.54 24.40 26.32
12 13 14 15
31.42 32.75 33.57 35.14
Name of the compound 5-Hydroxymethyl furfural 4-Hydroxy-3-methyl acetophenone Iberin nitrile Ethanone, 1-(3,4-dimethoxyphenyl)1,2-Benzene dicarboxylic acid, bis(2methylpropyl) ester n-Hexa decanoic acid 9,12-Octa decadienoic acid (Z,Z)cis-13-Eicosenoic acid Tetracaine Erucic acid 9-Octa decenoic acid (Z)-, 2-hydroxy-1(hydroxymethyl)ethyl ester γ-Tocopherol cis-13-Eicosenoic acid Stigmasterol Campesterol
(12.99), 5-Hydroxy methylfurfural (11.98). The peak area percent was moderate for cis-13-Eicosenoic acid (7.34) at the retention time of 32.75. 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl) ethyl ester (6.83), Campesterol (6.58), 9,12- Octa decadienoic acid (Z,Z)- (5.74), γ-Tocopherol (3.58). The peak area percent was low for Iberin nitrile (1.97), 1,2-Benzene dicarboxylic acid, bis(2-methylpropyl) ester (1.10), Whereas the peak area percent was very low 4Hydroxy-3-methyl
acetophenone
(0.58),
cis-13-
Eicosenoic acid (0.02) at the retention time of 21.17. The pharmacological activity of the identified compounds
were
discussed
below:
9,12-Octa
decadienoic acid (Z,Z)- were reported for their hypocholesterolemic,
5-alpha
reductase
inhibitor,
antihistaminic, insectifuge, antieczemic and antiacne.
Molecular Fomulae C6H6O3 C9H10O2 C5H9NOS C10H12O3 C16H22O4
MW
Peak area %
126 150 131 180 278
11.98 0.58 1.97 12.99 1.10
C16H32O2 C18H32O2 C20H38O2 C15H24N2O2 C22H42O2 C21H40O4
256 280 310 264 338 356
13.06 5.74 0.02 3.38 19.85 6.83
C28H48O2 C20H38O2 C29H48O C28H48O
416 310 412 400
3.58 7.34 4.99 6.58
n-Hexadecanoic acid for anti oxidant, hypocholesterolemic, nematicide, pesticide, lubricant, anti androgenic, flavor and hemolytic-5-α reductase inhibitor. Tetracaine also known as amethocaine was a potent local anasthetic of the ester group. It was mainly used in eye infection, antipruritic and spinal anesthesia. Erucic acid was applied in industrial process. Campesterol reduced the absorption of cholesterol in the human intestine (Choudhary and Tran, 2011). Campesterol was a precursor of anabolic steroid and boldenone. Boldenone undecylenate used to induce growth in cattle and abused anabolic steroids in sports. In 1950s it was reported for its lowering LDL, cholesterol level (Farquhar et al., 1958). Campesterol inhibited pro-inflammatory as well as matrix degradation mediators involved in osteoarthritis induced cartilage degradation (Reiter
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International Journal of Advanced Life Sciences (IJALS)
ISSN 2277 – 758X
Krishnaveni and Saranya, IJALS, Volume (9) Issue (2) May - 2016. RESEARCH ARTICLE
Fig. -1. GC-Chromatogram et al., 2007). γ-Tocopherol is anti-inflammatory in nature and used as a vitamin E supplement for its antioxidant activity (Ahmad and Ali, 2013). The results of chromatogram was shown in Fig.1. Fourier Transform Infrared Spectrophotometer Analysis The results of Fourier transform infrared analysis was depicted in Fig.- 2. The Fourier transform infrared spectrum identified the functional groups of the active components present in the Brassica hirta seed based on the peak values in the region of infrared radiation. When the sample was passed into the Fourier transform infrared spectrophotometer, the components functional groups were separated based on its peaks ratio. The infrared absorbtion below 1000 cm-1 corresponds to C-H bending vibrations. The absorption at 720.40cm-1 represented N-H group of primary, secondary amines,
Fig. -2 . Fourier Transform Infrared Analysis - result of Brassica hirta seeds by aldehydes, ketones and ester. The absorptions from 2800 - 2900 cm-1 were attributed to C-H stretching
while the absorption at 421.15cm-1 showed C-Cl stretch by alkyl halides. The absorbtions from 11501270cm-1 corresponds to the stretching vibrations of carbonyl C-O or O-H bendings, aromatic amines. The absorptions from 1500-1600cm-1 corresponded to the
vibrations specific to CH3, CH2 from lipids and methoxy derivatives as well as C-H aldehydes from cis double bonds. The absorptions from 3600 - 3350 cm-1 corresponded to the stretching vibrations of the – OH groups from water, alcohol, phenol,carbohydrate
aromatic domain and the N-H bending vibrations, C=O stretching mainly proteins. The absorptions from 1600 - 1760cm-1 corresponded to the bending
as well as from amides. The absorption at 3007.86 refered to C-H stretching of lipids. The absorption at 2924.94 cm-1 corresponded to C-H stretching of
vibrations of N-H from amino acids, C=O stretching
lipids and carboxylic acid. The peaks between 1020.34
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International Journal of Advanced Life Sciences (IJALS)
ISSN 2277 – 758X
Krishnaveni and Saranya, IJALS, Volume (9) Issue (2) May - 2016. RESEARCH ARTICLE - 1093.64 cm-1 represented primary amine C-N stretch. The peaks above 1500cm-1 formed a major peak and peaks below 1500 cm-1 formed a minor peak. The peaks revealed that Brassica hirta seeds contained compounds like alcohol, amides, amines, fluride, iodide, chloride, phosphine, bromide, sulfonates, aliphatic organo halogen, aliphatic and aromatic nitro compounds. The following peaks marked their respective compounds. The peaks at 3417.84 cm-1 showed the presence of compounds like alcohols, amines and phenols, 2924.94 cm-1 for amine salts, alkanes and 1746.10 cm-1 oleifinic (alkene), 1549.58 cm-1 aliphatic, aromatic nitro compounds, primary amines, 1461.00 cm-1 for aromatic compounds, 1160.37 cm-1 sulfur oxy compound, 1241.46 cm-1 aliphatic organo halogen for example flurides, aryl bromides, 3007.86 cm-1 for alcohol. From the reported datas, carboxylic acids and phenolic constituents exhibited bactericidal and fungicidal properties (Sultana et al., 2010; Soundararajan et al., 2012; Cowan et al., 1999). Conclusion Ethanol used for the extraction gave better extraction of phytochemical compounds. Phytochemicals present in Brassica hirta seed indicated that it could be a potential sources of active principle in the identification of novel medicines. Gas chromatography and mass spectrophotometry analysis confirmed to contain more peculiar compounds with good pharmacological activity. Fourier transform infrared spectroscopic study showed that Brassica hirta seed contained amines, carboxylic acid, and other aromatic compounds due to its pharmaceutical properties were described. The results of the present research could be extended depending upon their reported therapeutic uses in both animal as well as human models in order to find a better alternative for allopathy.
Acknowledgement The author wishes her thanks to Honorable Vice-chancellor Dr. C. Swaminathan, and Registrar Dr. M. Manivannan, for the administrative support and excellent infrastructure facilities provided and also thank Dean for Sciences Dr V. Krishnakumar, Professor and Head, Department of Physics, Periyar University, Salem, for giving permission to do FT- IR, and Co-ordinator, School of Bio-Sciences, Periyar University, Salem, and also Dr. J. Kalyanasundar, Assistant Professor cum Staff in charge for FTIR, Department of Physics, for performing FT-IR as well as Director Dr. K. Alagu-sundaram and Dr. S. Kumaravel, Senior scientist, Indian Institute of Crop Processing Technology, Thanjavur for analyzing the samples. The author would like to express her gratitude to her dedicated teachers. References Ahmad, B., and Ali, J. 2013. Physiochemical, minerals, phytochemical contents, antimicrobial activities evaluation and fourier transform infrared (FTIR) analysis of Hippophae rhamnoides L. leaves extracts. Afr. J. Pharm. Pharmacol., 7: 375 - 88. Balke, D. 2000. "Rapid aqueous extraction of mucilage from whole white mustard seed". Food Research International., 33: 347 - 356. Choudhary, SP. and Tran, L.S. 2011. "Phytosterols: Perspectives in human nutrition and clinical therapy". Current medicinal chemistry., 18: 4557 - 67. Cowan, M. M. 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev., 12 : 564- 82. Farquhar, John W., Sokolow, Maurice. 1958. Response of serum lipids and lipoproteins of man to beta- sitosterol and safflower oil. Circulation., 17 : 890 - 9.
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Krishnaveni and Saranya, IJALS, Volume (9) Issue (2) May - 2016. RESEARCH ARTICLE Gabay, O., Sanchez, C., Salvat, C., Chevy, F., Breton, M., Nourissat, G., Wolf, C., Jacques, C. and Berenbaum, F. 2010. "Stigmasterol: A phytosterol with potential anti-osteoarthritic properties". Osteoarthritis and Cartilage., 18 : 106–16.
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Soundararajan, V., Zakaria, Z., Chen, Y., Lachimanan, Y.L., Kanwar, J.R. and Sasidharan, S. 2012. The antimicrobial efficacy of Elaeis guineensis: characterization, in vitro and in vivo studies. Molecules., 17: 4860 - 77. Corresponding Author : M. Krishnaveni, Department of Biochemistry, School of Bio-Sciences, Periyar University, Salem – 11, Tamilnadu. Email :
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