Türkiye Kökenli Çörekotu Tohumlarının Antioksidan Kaynağı Olarak Değerlendirilmesi

Abstract

Doymamış yağ asitlerinin bir serbest radikal reaksiyonu üzerinden yürüyen otoksidasyonunu en engellemek için en çok kullanılan yöntem, yağlara ve yağ içeren gıda maddelerine antioksidan ilave etmektir. Yağların oksidasyonu sadece gıdaların bozulmasına değil, aynı zamanda canlı organizmada yaşlanma, kalp hastalıkları ve kanser gibi olumsuz etkilere de neden olur. Günümüzde yağlara ve yağlı maddelere antioksidan olarak çok bütillenmiş hidroksianisol, bütillenmiş hidroksitoluen gibi sentetik fenolik maddeler ilave edilmektedir. Ancak son zamanlarda bu aromatik yapıdaki maddelerin muhtemel olumsuz etkileri hakkında tüketicilerde bir tereddüt oluştuğundan, doğal antioksidanlara ilgi artmıştır. Bu bağlamda pek çok bitki ve tohumdan elde edilen ekstraktların antioksidan aktiviteleri saptanmış ve bunların arasında biberiye ve adaçayının ticari üretimleri başlamıştır. Bu çalışmada ise çörekotu tohumu, antioksidan kaynağı olarak incelenmiştir. Çalışmaların sonunda, antioksidanları ekstrakte etmek için önce çörekotu tohumlarındaki yağın giderilmesi gerektiği anlaşılmıştır. Ekstraktların antioksidan aktivitelerinin saptanması için kullanılan hızlandırılmış oksidasyon testlerinin tekrarlı ve güvenilir sonuçlar vermesi için hem oksidasyon koşullarının hem de kullanılan yağların tamamen aynı olması gerektiği de bu çalışmada elde edilen sonuçlardan biridir. Doğal antioksidanlann ekstraksiyonu için kullanılan üç çözücüden, metanol, % 96 'lık etanol ve SC-CCVden gerek renk gerekse polifenolik madde içeriği yönünden en uygun ekstraktı SC-CO2 vermiştir. Ayrıca çörekotu tohumlarının depolanmaları sırasında antioksidan aktivitelerini kaybettiği ve taze tohumlar ile çalışıldığında, ticari sentetik antioksidanlardan daha etkili doğal antioksidanlar elde edilebileceği de saptanmıştır.

Lipids that are rich in polyunsaturated fatty acids play an important role in human health and nutrition. The autoxidation of polyunsaturated fatty acids in fats and oils leads to the formation of hydroperoxides, which can damage the quality of foods and are considered to be detrimental to the health. Degradation of highly unsaturated fatty acids, via a free-radical chain mechanism, results in changes in odor and flavor of fats and oils and/or lipid containing foods. The steps of mechanism are depicted in Figure 1. H H H I I I -c-c=c- oxidation site tn oil molecule (RH) -H- H H H I I I -c-c=c- +O2 J fatty free radical (R) HHH -c-c=c- I 0 I o V. + H- J peroxide free radical (ROO) HHH -C-C=C- I o I o V H j hydroperoxide (ROOH) Figure 1. Free radical Oxidation of Vegetable Oil. The first step in free-radical chain mechanism is the formation of a fatty free radical. This occurs when hydrogen is lost from the ot-methylenic carbon in the fatty acid group. The fatty free radical is readily suspectible to attack by atmospheric oxygen, resulting in the formation of peroxides and hydroperoxides. As a final step, hydroperoxides may split into smaller organic compounds, such as aldehydes, ketones, alcohols and acids, which give the obnoxious odors and flavors characteristic of oxidative rancidity in vegetable oils. EX Antioxidants are used to preserve lipids and lipid-containing foods, by retarding rancidity, discoloration or deterioration due to autoxidation. Hence, the addition of antioxidants to foods has become increasingly common as a means of increasing the shelf-life and improving the stability of lipids and lipid-containing foods. Ingold classified all antioxidants into two groups, namely primary or chain- breaking antioxidants, which can react with lipid radicals to convert them to more stable products and secondary or preventive antioxidants which reduce the rate of chain initiation by a variety of mechanisms. Primary antioxidants are those substances which function by inhibiting or interrupting the free radical mechanism of glyceride autoxidation. Their ability to do this is based on their phenolic structure or the phenolic configuration within their molecular structure. As depicted in Figure 2 in a very simplified fashion, the primary antioxidant or phenolic substance functions as a free radical acceptor, thus terminating the oxidation at the initiation step. The antioxidant free radical which forms is stable and importantly, will not propagate further oxidation of the glyceride. R- + f r\ i ? RH + fatty phenol oil antioxidant free radical free radical molecule (stable resonance hybrids) Figure 2. Phenolic antioxidant mechanism in vegetable oil. Synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) are phenolic antioxidants with only one hydroxyl group in their chemical structures, act as free radical scavengers or chain-breakers. Synthetic antioxidants are widely used because of their high stability, low cost and efficacy. However, the safety and toxicity of them have been important concerns. Naturally occuring antioxidative components in foods include flavanoids, phenolic acids, lignans, terpenes, tocopherols, phospholipids and polyfunctional organic acids. Flavanoids are phenol derivatives synthesized in substantial amounts and widely distributed in plants. They can be subdivided into six classes of flavanoids and flavanoid-related compounds: flavones, isoflavones, flavonols and anthocyanins Nigella sativa L., also known as black cumin or fennel flower, is a member of the Ranunculaceae family and native to some parts of the mediterranean region. The seeds, on account of their aromatic nature, are used as condiment or spices in cooking, particularly in Italy, Southern France, India, Turkey and also as a carminative and diureetic by the Egyptians. The proximate composition of Nigella sativa L. seeds of Turkish origin has been determined to be as shown in Table 1. Table 1. Composition of Turkish origin Nigella sativa L. seeds Nigella sativa seeds contain also 0.5 to 1.5 % of essential oil, composed mainly of p-cymene, d-timonene, carvon, d- citronellol, citronelly acetate. Nigella sativa seed oil is typical of the oleic-linoleic group of vegetable oils, since those fatty acids comprise almost 80% of the total fatty acids. On the other hand, Nigella sativa seed oils, unlike the majority of other conventional vegetable oils, show a highly variable content of free fatty acids. This is due to the very high lipase activity in the seeds. In the Nigella sativa seed oil of Turkish origin, p-sitosterol was found to be the dominant sterol (69.4%); % of campesterol and stigmasterol were 11.9% and 18.6% respectively. The seed oil was also rich in polyphenols (1744 p.g/g). The investigations on the chemical compositions of Nigella sativa seed and seed oil showe that the seeds might have a high potential of antioxidant aactivity which has not been reported elsewhere. The purpose of the present investigation was to determine the extent of antioxidant activity of Nigella sativa seed of Turkish origin. In this study, samples of Nigella sativa seeds were purchased from a local market in Istanbul. The seeds used in this study ground in a Moulinex coffee grinder and selected (+300p. ; -850n) of ground seeds. Chemical composition of the seeds was determined according to the standart AOCS methods. Moisture content was found by oven-drying at 105°C. Total nitrogen content was determined using the standart Kjeldahl method. Crude protein was expressed as 6.25x N. crude fiber was determined by calculating the loss in weight of dried residue remaining after digestion of a fat-free sample with 0.25 M H2SO4 AND 0.6 M NaOH under specified conditions. Ash content was determined by incineration of the sample in a muffle furnace at 600°C for 2 h. Total fat content was obtained by subtracting the sum of protein, fat, ash and moisture from 100. XI The chemical composition of seeds determined according to the AOCS Methods was found as follows Table 2. Table 2. The chemical composition of the Nigella Sativa seeds used in the study According the Table 2., the oil content of the seeds are different. This is due to climate conditions. It is clear that the antioxidant activity changes in seeds growth in different places. In this study, it is not invesitgate seed type, it is examined the most suitable extraction conditions. 25 grams of ground seed or unfatted ground seed extracted with 50 ml of solvent for one and a half hour at room temperature. After filtering, the residues were extracted with 25 ml of solvent for 30 min. and filtered again. Combined filtrates evaporated in a rotary evaporator to 100 ml. 10 ml of this mixture was taken and evaporated up to a constant weight. SC-CO2 extraction was carried out in a pilot-plant super critic extractor. For this purpose, approximately 1.5 grams groun and defatted seeds extracted with CO2 for one hour at 40°C and 300 atm. The meals by extracting with CO2, collected in ethanol 96 %. And extract is up to 100 ml with ethanol. The antioxidant activites of the extracts were determined in accelerated oil oxidation which was examined by peroxide value. The antioxidant activities of extracts were compared with BHT, in several oils which include no antioxidant called control. The methanol extract from defatted seeds showed the better antioxidant acitivity than the other extracts which is first extracted with water and later extracted with methanol and directly extracted with methanol. However, each exract showed the antioxidant activity. That's why, it was decided to use defatted seed for antioxidant extraction through the study. It is examined antioxidant activity of seeds by using different solvents as follows in Table 3. The antioxidant activity is tested accelerated oxidation at 70°C in oven. According the results of this test, the ethanol extract showed higher antioxidant activity than methanol extract did. This may be due to solving different polyphenolics each other. However, both of them showed stronger antioxidant activity than BHT. The performance of SC-CO2 extract is given in the Figure 3. In the case, SC-CO2 extract is showed antioxidant activity but it is not as much as methanol extract did. It may be due to their polyphenol content is high and it may be act as proxidant. For this reason, it is tested at the less quantity SC-CO2 extract. In this test, 50 ppm extract showed antioxidant activity at the initation of oxidation but later it lost their effect. It also is differenlutesults between the tests at the same samples it may be due to different properties of the oils for example, different waiting time, production etc. The total polyphenolic contents of seed extracts were tested by Method (9. 1 10) of the AOAC. Briefly, 1 ml seed extract was added into a 100 ml volumetric flask xn containig 75 ml distilled water. S ml Folin Ciocalteau reagent and 10 ml saturated sodium carbonate solution were added to the flask and diluted to 100 ml with distilled water. The mixture was then shaken for 1 min and allowed to stand at room temperature for 30 min. The absorbance of the solution was wieved in a spectrophtometer at 760 nm. A standard curve was prepared with caffeic acid at concentrations ranging from 10-100u.g. The quantity of total polyphenolics contents of Nigella sativa seeds extracts by using different solvents are given in Table 3. The most total polyphenolic content is for SC-C02 extract which is also colourless extract. Thus, the most suitable solvent for antioxidant extraction is SC-CO2. Table 3. Total polyphenolic contents and total soluble substance of Nigella sativa seeds extracts by using different solvents The polyphenolic content and antioxidant activity of Nigella sativa seeds which were grown different regions in Turkey were tested in the other study. The chemical composition and phenolic content of these seeds are very different as follows in the Table 4. The difference in phenolic content causes showing different antioxidant activities. Seed II contains the less phenolic content than the others. It may be due to long storage time. Defatted seeds were extracted with methanol and tested antioxidant activities. The antioxidant activities of these extracts are given in Figure 4. The fresh seeds showed the better antioxidant activity than BHT. As a result of the properties of seeds like freshness and origin seed are very important to decise using as antioxidant source. Table 4. The chemical composition of the Nigella sativa seeds which were grown different regions in Turkey xni time (days) Figure 3. The peroxide number of sunflower oils treatment different solvent extracts 160 140 120 100 £ 80 ?Kontrol ?BHT -K.Maras -Çukurova ?Antakya Figure 4. The peroxide number of sunflower oils treatment different origin seed extracts.