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Our Research work on Isolation and Determination of Chemical components from the seeds of Pangium edule

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  • 1.ISOLATION AND DETERMINATION OF CHEMICAL COMPONENTS FROM SEEDS OF PANGIUM EDULE REINW.Jack Liam, I. Faridah-Hanum, Khalid Rehman HakeemFaculty of Forestry Universiti Putra Malaysia

2. 1. INTRODUCTION Pangium edule which is locally known as 'kepayang, as well as panggi ' belongs to the family Flacourtiaceae.These are big trees reaching heights of up to 40m, found wild or more commonly cultivated especially in villages in Sabah and Sarawak (Faridah Hanum, 1996). In Sabah and Sarawak, P. edule seeds are often sold in the markets for the preservation of fish or meat and as a vegetable after proper treatment. The oil obtained from seeds is used widely by the local communities in Sabah and Sarawak as cooking oil, although coconuts are sufficiently plentiful. The present investigation into P. edule was initiated following the observation of the wide use of its seeds as a preservative for fish and meat throughout the Dusun community in Sabah and the widely used oil from the seeds for cooking in Sarawak. 3. 2. METHODOLOGY The voucher specimen (FHI 400) was deposited in the Herbarium, Faculty of Forestry, Universiti Putra Malaysia.Prior to extraction, seeds were air dried after the mucilage and testa were removed.These were then ground to prepare for extraction with the chosen solvent. 800 g of P. edule was extracted by a cold soaking process (Harborne, 1973).The sample was soaked in petroleum ether for 3 days at room temperature using the separation funnel.The process was repeated thrice and each time the extracted residue sieved. The sieve was thickened by rotavapouring at a temperature of 45o C.The residue was labelled as PE 1. The remainder of the petroleum ether extraction was left to dry. The residue was then extracted using chloroform. The latter was labelled as PE 2. 4. 2.2 Biological Activity Test Different concentrations of PE 1 and PE 2 samples used in the Brine Shrimp Lethality test. The eggs of brine shrimp, Artemia salina were used which would hatch into tiny larvae (nauplii) when placed in brine, and swim towards a light source. The surviving shrimps were counted and Lethality Concentration at 50% (LC-50)with the degree of confidence at 95% for samples PE 1 and PE 2 was determined by the Finney Computer Programme. The LC50 value is part per million concentration of sample that kills the shrimps in brine.2.3 Component Separation by Thin Layer Chromatography (TLC) TLC plates were prepared according to Stahl (1969). Rf values of the spots were then determined to show the number of chemical components present in the samples where:Rf = Distance travelled by the spot centre of solute Distance travelled by solvent front 5. 2.4 Component Separation by Column chromatography As per Markl and Schmid (1975). (modified)Silica gel 60 (230-400 mesh) was used as the stationary phase and different ratios of mixed petroleum ether-chloroform as the mobile phase.2.5 Separation by Gas Chromatography In Gas Chromatography, samples PE 1 and PE 2 were first dissolved in chloroform and the separation was carried out on Carbowax 20M (25m) column.The peaks were then compared with the calibrated standard solution. Detection of components was carried out on the Flame Ionization Detector (FID).2.6 Identification by Gas Chromatography-Mass Spectroscopy All samples were subjected to GC-MS. The separation was carried out on C20X 25Mx. 25mm column.The identity of the components was confirmed by comparing their molecule mass spectrum with the Library search of file (Chem Database-Wiley I). 6. 3. RESULTS AND OBSERVATIONS 3.1 Table 1 shows the characteristics of the extract residue, PE 1 and PE 2. Residue Petroleum ether(PE 1) Chloroform (PE 2)Weight(g) 16.77 77.36Percentage 2.09 9.62Colour Yellow Brown3.2 Biological Activity Test: The biological activity test was carried out to determine if components of P. edule seeds were toxic to the living organism. LC50 value for PE 1 is 55758080 ppm and PE 2 is 94866880 ppm. It was found that both PE 1 and PE 2 samples were not very toxic. However, from the LC50 value, PE 2 is more toxic than PE 1. 7. 3.3 Component separation by Thin Layer Chromatography (TLC) The best solvent system for PE1 was 9:1 petroleum ether and acetone while the best solvent system for PE2 was 7:3 petroleum ether and chloroform.The best solvent system was determined by the best separation observed in the different ratios. From TLC, there are three components present in both Petroleum ether (PE1) and Chloroform (PE2) extraction. Results of the TLC: PE1 sample show that there were 3 spots determined, referred to as A1(Rf=0.56), A2 (Rf=0.25) and A3 (Rf=0.12). PE2 sample shows 3 spots to be present, referred to as B1 (Rf=0.56), B2 (Rf=0.21) and B3 (Rf=0.11). 8. 3.4 SEPARATION BY COLUMN CHROMATOGRAPHY (CC) AND GAS CHROMATOGRAPHY(GC) STUDIES By using a mixture of petroleum ether and chloroform as the mobile phase, the three major components separated were subjected to CC. From GC studies indicates that there are 3 major peaks representing 3 major components isolated from PE1 and PE2 samples. Extract ResiduesExtract Residues PE3 PE4B2, B3PE5 PE6 PE7 PE8 PE9Table 2.Major components A1 A2 A3 B1 B2 B3 Minor Components B2*B2, B1*, B3* B2*, B3* B2*, B3* B2*, B3* B2PE1PE2Retention times (Min.) 35.04 49.25 53.66 35.02 49.15 53.57 45.66 49.15, 54.3245.66, 33.20,53.11 45.66, 53.11 45.66, 53.11 45.66, 53.11 45.72It was observed that, separation and determination of components by CC and TLC are difficult because the components are colourless. GC however is a more viable method to adopt in determining the components present in P. edule. 9. 3.6 GAS CHROMATOGRAPHY-MASS SPECTROSCOPYDetermination of A1 component: The highest intensity (100%) was shown by m/z =73. Based on the molecular mass comparison of 256 and the library search of the file (Chem Database-Wiley-I) data, it was found that the probability of the component A1 is Hexadecanoic acid, (=Palmitic acid) which have the same molecule mass. Hexadecanoic acid or palmitic acid has been reported to be present in almost every vegetable and animal fat. Industrially, Palmitic acid is used principally in the form of commercial stearic acid which is a mixture of stearic and palmitic acids, containing 55% of the latter. In commercial pure form it has a variety of applications. Including the preparation of esters. Metallic salts, palmityl, alcohol, amides, nitriles, amines, and quarternary of ammonium salts. The acid or its derivatives find the use in the manufacture of synthetic detergents, soaps, cosmetics, greases, plastics and various types of protective and decorative coatings. It is suggested here that the Pangium edule seeds could be one of the future local sources of palmitic acid 10. Determination of A2 component: The highest peak intensity was shown by m/z = 55. Based on the molecule mass fragmentation comparison, it was found that A2 component almost has the same proton fragmentation as the Heptadecene-(8)-Carbonic-(1) acid or 8-Heptadecenoic acid from the library data. Besides, both components have the same molecule mass, 268. The characteristics, uses and potentials of 8Heptadecenoic acid are under continuing research nowadays. 11. Determination of A3 component: The highest peak intensity (100%) was shown by m/z = 67. Based on the comparison between molecule mass spectrum for A3 and the library data, it was found that proton fragmentation for A3 is almost the same as 9,12-Octadecadienoic acid ( Linoleic acid ). Besides, both have the same mass component of 280. It can be concluded that by comparing with the library data, A3 component is 9,12-Octadecadienoic acid with 90% probability. Linoleic acid is widely distributed in the plant kingdom.By frequency and proportionality, it is the most important polyalkenoic acid found in vegetable fats. It is characteristic and often forms the major component important in many commercialized fats such as cotton seed, soybean, peanut and sunflower seeds.Linoleic acid is almost invariably accompanied by oleic acid in vegetable oils and it is generally considered characteristic of semidrying and drying oils 12. Determination of B1 component: The highest peak intensity is shown by m/z = 60. Comparing the molecule mass fragmentation between B1 and library data indicates that both have the same proton fragmentation with Hexadecanoic acid. The mass molecule for both components is 256. Based on the comparison of the library data (Chem DatabaseWiley I) the probability of B1 component being Hexadecanoic acid is 96%. Determination of B2 component: The highest peak intensity is shown by m/z = 55. It was found that the fragmentation of proton for B2 component is almost the same as the Heptadecene(8)-Carbonic acid and both have a molecular mass 282. It can be concluded that B2 is Heptadecene-(8)-Carbonic acid. However, the pure component should be further studied. Determination of B3 component:The highest peak intensity was shown by m/z = 67. It shows that 9,12Octadecedienoic acid is the component that has almost the same proton fragmentation with the B2. Both also have the same molecule mass, 280. Based on the library data comparison, 99% probability of B3 component is 9,12Octadecadienoic acid. The pure component should however be further studied. 13. 4. Conclusion The present investigation into Pangium edule was initiated following the observation of the wide use of its seeds as a preservative for fish, meat and vegetables in Sabah and Sarawak. Two extract residue, PE 1 (Petroleum ether) and PE 2 (Chloroform) were collected and further determined. TLC, Column chromatography as well as gas chromatography showed 3 major peaks representing 3 major components isolated from PE1 and PE2 samples respectively. Using Gas Chromatography-Mass Spectroscopy (GC-MS), three components from the seeds namely Hexadecanoic acid (=Palmitic acid). Heptadecene-(8) Carbonic acid and 9, 12- Octadecedienoic acid (=Linoleic acid) were identified. Unlike palmitic an