Analytical characterization of fatty acids composition of datura alba
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Analytical characterization of fatty acids composition of datura alba Analytical characterization of fatty acids composition of datura alba Document Transcript

  • Analytical Characterization of Fatty Acids Composition of Datura alba Seed Oil by Gas Chromatography Mass Spectrometry Muhammad Nasimullah Qureshi,* Muhammad Siddique, Inayat-ur-Rahman and Farina Kanwal Medicinal Botanic Centre, PCSIR Laboratories Complex Peshawar, Pakistan Received July 9, 2010; Accepted November 23, 2010; Published Online December 14, 2010 Methyl ester derivatives of fatty acids were analyzed for the determination of the constituents of Datura seed oil. Gas chromatography coupled to mass spectrometer was used for these analyses. Results delivered that there were saturated as well as unsaturated fatty acids in Datura alba seed oil. Total of 15 different fatty acid components were identified and quantified. Methyl linoleate was found in highest con- centration (16.22%) among the identified analytes of interest. In addition methyl esters of Palmitic acid (6.59%), Oleic acid (5.41%) and Stearic acid (1.35%) were found. Concentrations of rest of the detected fatty acids were less than 1%. From the literature it appears that no such work has been performed for the determination of fatty acids in Datura alba seed oil. Keywords: Datura alba; Fatty acids; Methylation; GC-MS. 1. INTRODUCTION Datura alba (Family; Solanaccae) grows in warmer parts of the world particularly in south and south east Asia including Pakistan, India and Sri Lanka. This annual herb is bushy, smooth, fetid, 0.5 to 1.2 m in height also attaining 6 feet or more in rich soils. The leaves are 18 cm long and the flowers are white in colour.1,2 Besides its familiarity for toxicity and poisoning,3 it has uses for a number of diseases like asthma, muscle spasm, whooping cough, skin ulcer, hemorrhoids, anti-rheumatic etc. Its oil based preparation is used for healing of all types of wounds in Ayurveda and Siddha practice of medicine since long ago.4 Datura spe- cies produces a number of small seeds encapsulated in an apple like fruit capsule; hence the name “thornapple” is based on this fact. The most common and medicinally im- portant tropane alkaloids such as hyoscyamine, atropine and scopolamine have been isolated from Datura species. Clarification of the dual effect of this plant requires exten- sive research for the exploration of chemistry and pharma- cology of the plant under investigation. The present work is a part of these investigations focusing on the determination of fatty acids in oil extracted from Datura seeds. Due to biological importance5,6 fatty acids have gained importance in food nutrition evaluation7-10 and in the diag- nosis of certain diseases and pharmacology.11 Fatty acids with unsaturation, either monounsaturated or polyunsatu- rated, have been used in lowering the risks of heart disease, against inflammation and in enhancing the immunity or im- mune system.12-17 A number of analytical techniques have been applied for the determination of fatty acids. These include: enzy- matic, spectrophotometric, HPLC18-20 and gas chromatog- raphy (GC).21-23 GC-MS is the method of choice for the analysis of fatty acids due to various reasons like speed, resolutions and sensitivity.24,25 From the literature it ap- pears that most of the research work performed is the deter- mination of alkaloids which are main constituents of this genus. No remarkable work has been carried out for the de- termination of fatty acids in Datura alba seed oil which is needed in order to explore its pharmacological importance. 2. RESULTS AND DISCUSSION Table 1 summarizes the results obtained from the GC- MS analysis showing the relative concentration of individ- ual esterified fatty acids based on the external standard method and the standard deviation values among the three results in each case. Analyses were performed three times and the values of area and concentration in Table 1 are the average of three measurement results. Quantification of FAMEs was performed using three points calibration curve Fatty Acids in Datura alba Seed Oil by GC-MS J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 1 * Corresponding author. Tel: 0092-91-9216240-43; Fax: 0043-512-5072767; E-mail: mnasimuq@yahoo.com
  • with R2 value less than 0.99 (R2 > 0.99) in each case. Fig. 1 shows the GC-MS chromatogram obtained from fatty acid standard mixture of 37 components while Fig. 2 is the GC- MS chromatogram of Datura alba seed oil with properly labeled signals of analytes detected. Both the saturated and unsaturated fatty acids were found in the sample under in- vestigations. Linoleic acid was found in highest concentra- tion which is necessary for the maintenance of growth. It has been shown to be a potent inhibitor of cyclooxiginase-2 (COX-2) catalyzed prostaglandin biosynthesis.29,30 Among the other fatty acids with concentrations more than 1% are: Palmitic acid (6.59%), Oleic acid (5.41%) and Stearic acid (1.35%) were found. Amount of rest of the fatty acids were less than 1% (Table 1). From the results it is clear that Datura alba seeds, be- sides its toxicity, can also be used in various pharmaceuti- cal products as it contains different bioactive compounds like fatty acids. The method applied is a reliable method of analyzing simultaneously many fatty acid components in a single run. 3. EXPERIMENTAL 3.1. Chemicals and reagents Boron triflouride solution in methanol (10%) was purchased from Fluka Chemie (Buchs, Switzerland). So- dium hydroxide solution (methanolic; 0.5 N) and sodium chloride (analytical grade) were obtained from Merck (Darmstadt, Germany) while methanol (HPLC grade), n-hexane (HPLC grade) were from Fischer Scientific (Leicestershire, UK). Helium gas (99.9999%) from Pak gas (United Arab Emirates) was procured. Tridecanoic acid methyl ester and Fatty acid methyl esters (FAMEs) 37 com- ponents standard mix were obtained from AccuStandard (Newhaven, Connecticut USA). These 37 components are: methyl ester of hexanoic acid, caprylic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, myristoleic acid, pentadecanoic acid, pentdecenoic acid, palmitic acid, palmitoleic acid, margaric acid, hep- tadecenoic acid, stearic acid, oleic acid, elaidic acid, octa- decenoic acid, linoleic acid, octadecadienoic acid, g-lino- lenic acid, linolenic acid, arachidic acid, eicosenoic acid, eicosadienoic acid, 8,11,14-eicosatrienoic acid, heneico- sanoic acid, arachidonic acid, eicosatrienoic acid, eicosa- pentaenoic acid, behenic acid, eruccic acid, docosadienoic acid (C22:2), tricosanoic acid, tetracosanoic acid, docosa- hexaenoic acid and tetracosenoic acid. Deionized water was used through out the experimental work. 3.2. Preparation of standard Internal standard was prepared by dissolving 13.7 mg of tridecanoic acid methyl ester in 1 mL hexane. External standard was prepared by diluting 10 mg of 37 component FAMEs mix standard to 10 mL with dichloromethane. From this solution further working standard solutions were prepared. 2 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 Qureshi et al. Table 1. Quantification results of fatty acid methyl esters # Name Retention time (min.) AreaØ Conc. (%)Ø Std. Dev.* 1 C6:0; Hexanoic acid, methyl ester 3.060 19654 0.16 0.002 2 C8:0; Caprylic acid, methyl ester 4.957 11442 0.11 0.003 3 C10:0; Capric acid, methyl ester 6.796 3454 0.02 0.002 4 C12:0; Lauric acid, methyl ester 8.552 2928 0.01 0.003 5 C14:0; Myristic acid, methyl ester 11.000 39973 0.18 0.010 6 C15:0; Pentadecanoic acid, methyl ester 12.670 2407 0.01 0.003 7 C16:0; Palmitic acid, methyl ester 14.692 2674581 6.59 0.020 8 C16:1c; Palmitoleic acid, methyl ester 15.213 8191 0.21 0.002 9 C17:0; Margaric acid, methyl ester 16.985 13220 0.09 0.003 10 C18:0; Stearic acid, methyl ester 19.704 427823 1.35 0.010 11 C18:1c; Oleic acid, methyl ester 20.266 816146 5.41 0.003 12 C18:1n8T; Octadecanoic acid, methyl ester 20.433 86541 0.92 0.004 13 C18:2c; Linoleic acid, methyl ester 21.912 1554375 16.22 0.002 14 C20:0; Arachidic acid, methyl ester 27.306 30982 0.22 0.004 15 C20:2; Eicosadienoic acid, methyl ester 29.756 22200 0.65 0.005 * Standard deviation values for the three measurement results; Ø Average of three measurement results.
  • 3.3. Extraction of oil and preparation of FAMEs About 100 g powdered seed material was extracted with 250 mL n-hexane26 for six hours through soxhlet ex- traction apparatus. The extract was concentrated by recov- ering the solvent using rotary evaporator. Fatty acids are polar compounds and are not volatile. For gas chromatographic analysis it is necessary that the sample to be analyzed must be volatile. In order to make fatty acids present in the oil volatile, derivatizaion is per- formed prior to GC-MS analysis. Methylation is the most general method of converting non-volatile fatty acids into volatile fatty acids methyl esters (FAMEs).27 Methylation of fatty acids was performed with BF3-methanol as deri- vatizing reagent, which is the most accepted procedure for converting fatty acids into FAMEs.23 Derivatization was performed according to the AOAC standard reference method.28 To a known amount of sample (equivalent to 25 mg fat) was added 0.1 mL internal standard (1.37 mg) and 1.5 mL of sodium hydroxide solution in methanol (0.5 N), sealed and heated in boiling water bath for 5 minutes. The hydrolyzed sample was cooled and added 2.5 mL of boron triflouride solution in methanol (10%). The solution was then sealed and heated in boiling water bath for 30 minutes and cooled. To the esterified solution was added 5 mL satu- rated sodium chloride solution and extracted twice with 1 mL hexane. The hexane extract was filtered through 0.45 mm membrane filter and injected 1 ml to GCMS using auto injector system. 3.4. Chromatographic separation of FAMEs Agas chromatograph from Shimadzu hyphenated to a mass spectrometer QP 2010 plus (Tokyo, Japan) equipped with an auto-sampler (AOC-20S) and auto-injector (AOC- 20i) was used. Helium was used as carrier gas. All chro- matographic separations were performed on a capillary column (TRB-FFAP; Technokroma) having specifications: length; 30 m, i.d.; 0.35 mm, thickness; 0.250 µm, treated Fatty Acids in Datura alba Seed Oil by GC-MS J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 3 Fig. 1. GC-MS chromatogram of 37 components standard. Chromatographic conditions: inj. vol.: 1 µL, carrier gas: Helium, column: TRB-FFAP capillary column (length; 30 m, i.d.; 0.35 mm, thickness; 0.250 µm, treated with polyethylene glycol), MS scanning: 85-380 m/z. View slide
  • with polyethylene glycol. Other GC-MS conditions are: ion source temperature (EI); 250 °C, interface temperature; 240 °C, pressure; 100 KPa, solvent cut time; 1.8 min. 1 µL of sample and standard were injected into the GC column. Injector was operated in a split mode with a split ratio 1:50. Injection temperature was 240 °C. The column temperature program started at 50 °C for 1 min and changed to 150 °C at the rate of 15 °C/min. The temperature was raised to 175 °C at the rate of 2.5 °C/min and hold for 5 minutes. Then the temperature was increased to 220 °C at the rate of 2.5 °C/min and kept constant for 3 minutes. Total elution time was 43 minutes. MS scanning was performed from m/z 85 to m/z 380. GC-MS solutions software provided by the sup- plier was used to control the system and to acquire the data. Identification of the compounds was carried out by com- paring the mass spectra obtained with those of standard mass spectra from the NIST library (NIST 05). REFERENCES 1. Nadkarni, K. M. Dr. KM Nadkarni’s Indian Materia Medica; Popular Prakashan: Bombay, 1994. 2. Kuganathan, N.; Saminathan, S.; Muttukrishna, S. Internet J. Toxicol. 2008, 5(2). 3. Steenkamp, P. A.; Harding, N. M.; Heerden, F. R. v.; Wyk, B. E. v. Forensic Sci. Int. 2004, 145, 31-39. 4. Priya, K. S.; Gnanamani, A.; Radhakrishnan, N.; Babu, M. Journal of Ethnopharmacology 2002, 83, 193-199. 5. Wallace, F. A.; Neely, S. J.; Miles, E. A.; Calder, P. C. Immunol. Cell. Biol. 2000, 78, 40-48. 6. Cherif, S.; Frikha, F.; Gargouri, Y.; Miled, N. Food Chem. 2008, 111, 930-933. 7. Tomaino, R. M.; Parker, J. D.; Larick, D. K. J. Agric. Food Chem. 2001, 49, 3993-3998. 8. Skonberg, D. I.; Perkins, B. L. Food Chem. 2002, 77, 401-404. 9. Martin, C. A.; Carapelli, R.; Visantainer, J. V.; Matsushita, M.; de Souza, N. E. Food Chem. 2005, 93, 445-448. 10. Philip, C. C. Prostaglandins, Leukot. Essent. Fatty Acids 2008, 79, 101-108. 11. Stoddart, L. A.; Smith, N. J.; Milligan, G. Pharmacol. Rev. 2008, 60, 405-417. 12. Calder, P. Lipids 1999, 34, S137-S140. 4 J. Chin. Chem. Soc., Vol. 58, No. 1, 2011 Qureshi et al. Fig. 2. GC-MS chromatogram of Datura alba seed oil. Chromatographic conditions: as in Fig. 1. View slide
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