ANALYSIS ON SOME LEVEL OMEGA 3 EXTRACT DARA SHELLS (Anadara Granosa) USING GAS CHROMATOGRAPHY

A. Muflihunna Fakultas Farmasi, Universitas Muslim Indonesia E-mail: Amchund124@gmail.com

ABSTRACT Has done research on the analysis of the levels of Omega-3 fatty acids and chemical physics analysis of extracts of Dara shells (Anadara granosa) by gas chromatography. Dara shells (Anadara granosa) derived from Barranglompo Island in the city of Makassar . Dara shells dried clam meat in extraction by maceration method by using the solvent n - hexane, ethanol, ether for 9 days (every 3 days in the filter and replaced the solvent). Extraction results in saponification with NaOH - methanol , and then esterified with BF3 - methanol solvent . To produce pure ester extracted again with n - hexane ( pa ) and saturated NaCl . As much as 1 mL injected into a pure ester gas chromatography . Chemical analysis of the extract physics Dara shells is the percentage yield , water -binding capacity and water solubility index anderson method, the water content with the oven method, levels of total ash, crude protein by the Kjeldahl method, crude fat content by gravimetric method, and the method of reduction of carbohydrate content . Chemical physics analysis results show the value of yield of 24.35 % , water -binding capacity of 1.6248 g / ml , water solubility index of 0.202 g / ml , amounting to 79.0045 % moisture content , total ash content of 1.072 %, protein content coarse wet weight of 2.25 %, crude fat content of 8.47 %, carbohydrate content of 9.2035 %. Meanwhile, the percentage of the relative levels of Omega3 on virgin shells as much as 12.734 % of the weight of fat mussels virgin. Results of analysis of total levels of Omega-3 fatty acids in the form of shells virgin linoleta , arachidat acid , and acid Cis Eicosatrionat showed different results from the ether extract, hexane, and methanol in chromatography. Conclusion this study shows that the levels of Omega - 3 derived from Dara Shells can be measured by using the 3 kinds of solvents and measured by gas chromatography. Keyword : omega-3, Dara shell, gas chromatography, ether, hexane, methanol INTRODUCTION Background Linoleic acid is a fatty acid aesnsial , the fatty acids that can not be formed in the body, so it must be consumed through food. Linolenic acid is an unsaturated fatty acid plural ( polyunsaturated fatty acids , PUFA ) straight chain of 18 carbon atoms . Isome has two forms , namely -linolenic acid ( ALA ) and - linolenic acid ( GLA ) , which both have a carbon framework containing double bonds are not conjugated . In IUPAC name of the chemical structure of ALA is acid

9,12,15-oktadekatrenoat, while the GLA is acid 6,9,12 - oktadekatrienoat ( Fasya AG, et al., 2012 and Panagan AT, et al., 2011). lpha linolenic acid ( ALA ) is known omega - 3 fatty acids are known to have more efficacy than the other fatty acids, particularly in preventing damage to cell membranes . Besides useful for normal growth, omega- 3 also has a critical role in the formation and growth of brain function. Closer study also found that omega- 3 fatty acids can reduce inflammation and help prevent certain chronic diseases such as heart disease and arthritis ( Kataren S, 1986). Sea shells contain high nutritional composition such as protein, vitamins and minerals also contain unsaturated fatty acids omega-3. Benefits of consuming omega-3 fatty acids in the form of oil extracts or concentrates have been shown to affect the prevention and treatment of coronary heart disease. Many studies have been done abroad has been much use of marine products such as sea shells to prevent the occurrence of coronary heart disease. One type of sea shells as a potential source of our marine resources is Dara shells (Anadara granosa) . The chemical composition of virgin shells vary greatly depending on the species , sex , age , and habitat . Virgin shells containing 9-13 % protein, 0-2 % fat, 1-7 % glycogen and has a calorific value of 80 calories per 100 grams of fresh meat. Shellfish are also rich in virgin succinic acid , citric acid , glycolic acid. In addition, virgin shells contain omega- 3 fatty acids 1.608.55 mg / kg shellfish meat , EPA 320.03 mg / kg and 431.95 mg DHA / kg (Luigi C et al. 2005). Oils or fats contained in virgin shells can be classified into two based on the fatty acid saturation , namely saturated fatty acids and unsaturated fatty acids. The difference lies in the double bonds, saturated fatty acids have no double bonds , while unsaturated fatty acids containing double bonds. This difference causes the difference in physical and chemical properties of these two groups of fatty acids (Ackman, 1982). The main fatty acids of animals is configured omega3, whereas in plants containing fatty acids with omega - 6 configuration . Natural fatty acids including omega-3 fatty acids are linoleic, eicosapentaenoic acid ( EPA ) and dekosaheksaenoat acid (DHA). Consuming omega-3 fatty acids in amounts sufficient to reduce the cholesterol content in the blood and reduce the risk of

heart disease, as well artherosklerosis can selectively kill cancer cells and cure the symptoms of arthritis rheumathoid. Clinical effects of omega-3 fatty acids in lowering blood cholesterol levels may be related effect on the production of lipoprotein transport mechanism in the liver that are secreted into the blood . Cholesterol in the blood is essentially exists in the form of lipoproteins, namely Very Low Density Lipoprotein (VLDL), Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL). Unsaturated fatty acids omega-3 can inhibit the synthesis of VLDL and LDL production was reduced as a result. High levels of secreted VLDL and LDL cholesterol deposits in the blood rise, because VLDL and LDL are transport proteins that carry triglycerides, cholesterol and phospholipids from the liver to the rest of the network . Whereas HDL transports cholesterol to the liver, further broken down into bile acids and excreted through the body's excretion (Kinsella et al., 1990). Another favorable clinical aspects of consuming omega-3 fatty acids prevent the disease is atherosclerosis, thrombosis and arthritis. This is presumably due to the antagonistic nature of the omega-3 fatty acids can decrease the activity of conversion of linoleic acid into arachidonic acid, as well as the oxidative conversion of arachidonic acid into eicosanoids. In addition to omega 3, virgin shells are also rich in antioxidant compounds . One is the astaxanthin content of antioxidant compounds. Amount of antioxidant compounds derived from animal sources is very less but has a very big role . The surprising thing is that classified as carotenoids astaxanthin . According to experts , astaxanthin 1000 times more potent as an antioxidant than vitamin E. Shrimp, salmon, and shellfish are a potential source of astaxanthin. The antioxidant activity against lipid peroxide works and oxidation of LDL cholesterol and the dangers of UV rays, and helps eyesight, immune response, and reproduction (Murray RK., 2003). Based on the above description , it is important to research the alleged virgin shells containing omega 3 and antioxidant compounds. Both of these compounds are expected to lower high cholesterol levels . The results of this study can provide information on the composition of fatty acids in virgin shells, especially the potential of omega-3 fatty acids and antioxidant compounds in preventing coronary heart disease.

METHODS 1. Sampling Samples Dara shells (Anadara granosa) obtained from Lompo Barrang Island South Sulawesi city of Makassar. 2. Dara Shellfish Preparation Dara shells (Anadara granosa) opened its shell, meat, cleaned, and dried with aerated, then mashed. Dry powder Dara shells included in the beaker contains distilled water and heated until the volume is reduced by 50%. Results filtered and the filtrate concentrated heating at a temperature not over 75oC to obtain a dry extract. Next, the dried extract powder weighed to determine its rendamen. 3. Analysis of Chemical Physics Physics analysis procedures conducted on Dara shells are as follows: a. Determination of the yield of the Dara shells Rendamen Dara shells is calculated based on the dry weight. Dara shells yield can be calculated by the formula: Rendemen (%) = (weight of flour extracts mussels virgin) / (weight of flour Dara shells ) x 100% b. Water-binding capacity and water solubility index, method of Anderson (Paton and Spartt, 1984) A total of 2.5 grams of sample is introduced into a centrifuge tube known weight (t). A total of 15 ml of distilled water was added to the centrifuge tube and divortex that all samples dispersed in water. Tube filled with suspense and then weighed to determine the weight initially (at). After that, the tubes were centrifuged at 2000 rpm at room temperature for 15 minutes. Obtained supernatant was transferred into another container which has been known to weigh to determine the weight of the supernatant (b).A total of 2 ml of the supernatant was placed in a porcelain dish known weight (x). The cup is then heated in an oven at 105oC for one hour, and cooled in a desiccator and then weighed again (d). Water absorption index and water solubility index is determined by the following formula: Water absorption index (b / b) = (a)-b) / (weight of sample) Water solubility index (w / v) = (d-x) / Volume

4. Chemical analysis procedures were carried out on virgin shells (Anadara granosa) are as follows: a. Moisture content (AOAC, 1995) Water content measurements performed with the oven method. Porcelain cup dried in an oven at 100 C for 15 min, cooled in a desiccator to room temperature, then weighed (A). Balance sheet is clear and as many as 5 grams of sample is introduced into the cup and then weighed again (B). Dish containing the sample was dried in an oven at 100oC for 6 hours, cooled in a desiccator for 15 minutes and weighed until constant weight (C). Water content = (C-A) / (B-A) x 100% b. Levels of total ash (AOAC, 1995) A total of 5 grams of sample is introduced in a cup that has been burned in the furnace, cooled and weighed (Ws). Then the cup along with the sample was burned in a furnace to obtain the ashes. Incineration is done in two stages, first at a temperature of 400oC and the second at a temperature of 550oC. After that, the dish containing the sample was cooled in a desiccator and weighed (Wa). Levels of total ash = (Ws-Wa) / (sample weight) x 100% c. Levels of crude protein (Kjeldahl method) A total of 0.5655 and 0.5293 gram sample was weighed and put into tube kjeldahl. Added 1 gram of selenium and 20 ml of concentrated H2SO4. Samples in dekstruksi up colored clear solution. Once cool, dilute samples with distilled water into a 100 mL volumetric flask. A total of 5 ml flask contents Kjehldahl transferred into a flask and added 40% NaOH to do the process of distillation. 200 ml Erlenmeyer flask containing 20 ml of H3BO3 solution and 2-4 drops of methyl red indicator and the blue bromkresol, associated with distillation, distillation end of the channel must be submerged in a solution of H3BO3. Distillation is then performed to accommodated approximately 50 ml of distillate green erlenmeyer. Furthermore distillate titrated with 0.0142 N H2SO4 solution until the color changes to purple and blank titration was also performed. % N = ((mL-HCl-mL blank)) / (mg of sample) x N x 14.007 x 100% Description:% protein =% N 6.25

d. Coarse fat content (gravimetric method) Each sample weighed as much as 1.0218 and 1.0256 gram. Inserted into the sample 15 ml test tube, add 10 ml of chloroform. Chloroform meniscus surface marked with a paper label, then closed with a rubber stopper and then shuffled back, let it stay overnight. The next day if the volume is reduced chloroform can be added back by using a pipette. Shaken until homogeneous and filtered using filter paper into another test tube. Pipetted into 5 ml fill the cup that has been known weight (a gram). In oven at a temperature of 100o C for 3 hours. Inserted into eksikator 30 minutes and weighed (g b). Calculated fat content. "% Fat content" = (P (ba)) / (g sample) x 100% e. Carbohydrate content Calculation of carbohydrate content was conducted by the reduction (by different) as follows: Carbohydrate content (%) = 100% - (kadarair + protein + fat + ash)% 4. Saponification of Dara Shells Saponification process is intended to free fatty acids from the glycerol bonds so it will be easier for esterified. The process is ethanol extract of mussels virgin weighed as much as 0.5 grams put in a bottle and add 2 ml of NaOHmethanol and heated over a water bath (waterbath) for 10 minutes and cooled for 8 minutes. 5. Esterification using BF3-methanol from Dara Shells Esterification performed using BF3-methanol solvent to obtain the esters to be analyzed further by using a gas chromatography. The esterification process the samples that have been through the process of saponification was added 2 ml of BF3-methanol solvent, heated for 40 minutes and cooled for 8 minutes. Then added 4 ml of n-hexane (pa). Subsequently centrifuged for 3 minutes then added saturated NaCl solution and formed two phases. 6. Identification of omega-3 with gas chromatography The top layer is taken as the result 1L centrifugation using Syring then injected into the injector and kromatogramnya awaited results in the form of peakpeak.

RESULT The results of the analysis of chemical physics from virgin shells (Anadara granosa) derived from Makassar Barranglompo can be seen in Table 1.

Table 1: Results of analysis of chemical physics from Dara shells ( Anadara granos a) derived from Barranglompo Makassar. Analysis of chemical physics from Sample Rendemen Water-binding capacity Water solubility index Level of waterr Levels of total ash Levels of crude protein Coarse fat content Carbohydrate content Result 23,45 % 1,6248 gram/ml 0,202 gram/ml 79,0045 % 1,072 % 2,25 % 8.47 % 9,2035 %

Physics analysis procedure performed is the determination of the yield of the Dara shells (Anadara granosa ) aims to see what percentage yield of Dara shells, either as powdered or after passing through the heating process to obtain a dry extract. And yield results from Dara shells is 24.35 %. Chemical analysis include water binding indices obtained at 1.624 g / ml and water solubility index of 0.202 g/ml. Water-binding capacity and water solubility index using the Anderson method aims to obtain the results of how big the scallops Traffic virgin in binding water and the water level in the kelelarutan . Here the water binding capacity associated with the hygroscopic properties of a sample . Water solubility index associated with polarity type a sample of water. Subsequent chemical analysis is the determination of water content obtained from shellfish samples virgin amounted to 79.0045 % , ash content obtained amounted to 1.072 % , crude protein content is 1.88 % pa and 2.25 % dry weight wet weight , fat content rough obtained amounted to 8.47 % , and crude carbohydrate content of 9.2035 % is obtained.

Omega-3 fatty acids are unsaturated fatty acids that have double bonds plural many, the first double bond is located at the third carbon atom from the omega metal cluster , the next double bond is located at the third carbon atom of the double bond earlier. Unsaturated fatty acids are also known as essential fatty acids can be said to be essential karenatidak synthesized in the body, and therefore must be obtained from food, as well as minerals and vitamins. Dara shells (Anadara granosa) is one of the results of marine Indonesia that have high economic value and potential as a source of food to meet the Indonesian society source of omega-3 naturally. samples obtained at the fish auction Paotere in Makassar South Sulawesi Province. Analysis of the concentration ratio of omega 3 to use 3 types of solvents are ether, methanol, and n-Hesan done qualitatively and quantitatively using Gas Chromatography instrument. Analysis results can be seen in table 2 and figure peak chromatogram in Figure 3-5.

Table 2. Results of analysis of omega 3 in ethanol extract, ether and N-hexane Dara Shell (Anadara granosa) No 1. 2 3 4 Omega-3 Arachidat acid Linolenat acid Eicosatrinoat acid Eicosapentanoat acid Total Konsentrasi (%) ekstrak n-Heksan Eter 0,428 4,144 3,878 0,007 8,457 14,714 1,317 0,404 2,464 18,899

Etanol 9,019 0,442 3,273 12,734

The results showed that the content of omega-3 on virgin extension on the concentration of ethanol extract, ether and n-hexane have differences. Eicosapentanoat arachidat acid and acid showed high solubility in the solvent nhexane. Eicosatrinoat acid and linolenic acid showed high solubility in ether solvent. So that the overall solubility of the solvent n-hexane the most high at 18.899 %.

Table 3. Chromatogram peak outcome data on mussels Dara ether extract (Anadara granosa) using Gas Chromatography

From the results of the quantitative analysis of Dara shells using gas chromatography to measure the peak area . Retrieved levels of omega- 3 fatty acids a total of 8.457 % for 60 minutes consisting of fatty acids Arachidat of 0.428 % , linolenic fatty acid at 4.144 % , 11,14,17 Eicosatrionat Cis acid at 3.878 % and 0.007 % Eicosapentanoat acid to weight fatty acids . Table 4. Chromatogram peak outcome data on ethanol extract of mussels Dara (Anadara granosa) using Gas Chromatography.

Fatty acids derived from microorganisms that become food for shellfish . Water temperature is too low ( sub-tropical waters ) may increase fatty acid content in shellfish , plankton and algae because it can increase the solubility of oxygen that can accelerate the synthesis of fatty acids and the enzymes in desaturase reaction .

Table 5. Chromatogram peak outcome data on n-hexane extract of mussels Dara (Anadara granosa) using Gas Chromatography.

CONCLUSION The conclusion of this study is Dara shells containing polyunsaturated fatty acids omega-3 and omega-3 solubility differences in the three types of solution used. Concentration of n-hexane extract showed the highest value (18.899%) compared to the ether and methanol extracts.

BIBLIOGRAPHY Ackman, RG. 1982. Fatti Acid Compocition of Fish Oil. Dalam Nurtitional Evaluation of long Chain Fatty Acid in fish oil. Barlow S. M and M. E Stansby (Ed). Acad Press Ltd : London

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AOAC. 1995. Official Methods of Analysis of Official Analytical Chemistry. AOAC.Inc. Arlington. Fasya AG, et al., 2012. Isolasi asam-9z, 12z, 15z,-oktadekatrienoat dari biji selasih (Ocimum basilicum). Alchemy 2 (1): 1-11 Kinsella, JE., KS.Broughton and JW.Whelan. 1990. Dietary Unsaturated Fatty Acids Interaction and Posible Need in Relation to Eicosanoid Shyntesis, J. Nutrition Biochemistry (3) : 11-16 Luigi C, Tania G and Oscar R. 2005. Relationship between anti-oxidant capacity and manganese accumulation in the soft tissues of two freshwater molluscs : Unio pictorum mancus (Lamellibranchia, Unionidae) and Viviparous ater (Gastropoda, prosobranchia). J. Linmol 10: 34-441 Murray, R.K. 2003. Biokimia Harper. EGC : Jakarta Panagan AT., Heni Y., and Jojor UG. 2011. Analisis kualitatif dan kuantitatif asam lemak tak jenuh omega-3 dari minyak ikan patin (Pangasius pangasius) dengan metode kromatografi gas. Jurnal Perikanan 2: 23-30 Paton, D. and W. A. Spratt. 1984. Component interaction in the cooking process influence of process condition on the functional viscocity on the wheat flour system. Journal of food Science (49) : 1380-1385.

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