Changes in Fatty Acid, Tocopherol and Sterol Contents of Oils Extracted from Several Vegetable Seeds.

Oil contents of seeds changed between 15.89 g/100 g (purslane) and 38.97 g/100 g (black radish). Palmitic acid contents of oil samples were found between 2.2 g/100 g (turnip) and 15.0 g/100 g (purslane). While oleic acid contents of oil samples change between 12.1% (turnip) and 69.8% (purple carrot), linoleic acid contents of oils were determined between 8.9% (black radish) and 57.0% (onion). The highest linolenic acid was found in purslane oil (26.7%). While α-tocopherol contents of oil samples range from 2.01 mg/kg (purple carrot) to 903.01 mg/kg (onion), γ-tocopherol contents of vegetable seed oils changed between 1.14 mg/kg (curly lettuce) and 557.22 mg/kg (purslane). While campesterin contents of seed oils change between 203.2 mg/kg (purple carrot) and 2808.5 mg/kg (cabbage Yalova), stosterin contents of oil samples varied from 981.5 (curly lettuce) to 4843.3 mg/kg (purslane). The highest brassicasterin and δ5-avenasterin were found in red cabbage oil (894.5 mg/kg) and purslane seed oils (971.3 mg/kg), respectively. Total sterol contents of seed oils changed between 2960.4 mg/kg (purple carrot) and 9185.1 mg/kg (purslane). According to the results, vegetable seeds have different bioactive compound such as fatty acid, tocopherol and phytosterol.


Introduction
The most of natural crude oils has limited applications, although majority of seed oils have good structural chemical profiles 1,2 . Fatty acids, tocopherols, tocotrienols and sterols are important bioactive compounds of oil seeds. The chemical composition of vegetable oils, such as the composition of fatty acids, tocopherols and sterile glycoside contents, has a very important effect for industrial applications. The α-tocopherol among all forms of vitamin E plays the essential role in human nutrition and health. For instance, it protects unsaturated fatty acids against oxidation and ensures the stability of lipid membranes 3 5 . There is an important role of α-tocopherol at the preventing lipids and lipid containing foodstuffs from oxidation during storage in food production and very important biological activities, such as anti-inflammatory 5 . But, tocol-related compounds occur in seeds in variable amounts, and their biological activities changes between individual constituents 6 . Vegetable oils are important part of human livelihood all over the world. Due to ever diminishing sources of fats and oils, there is the search of new sources of oil 7 . Vegetable seed oils have shown to be major source of dietary ingredients related to their fatty acid composition 8 . Unconventional oilseeds are used for foods and cosmetics as medicinal purposes 8,9 . The seed and oil cake are a good fodder and energy for cattle, and their oils are used in cosmetics, pharmaceuticals, oleochemicals. These by-products contain some valuable substances such as fatty acids, tocopherols, sterols and sterylglycosides with potential applications in food industry mostly go to waste. Most seeds contain a significant amount of lipids, with omega-3-fatty acids accounting for about 60 of the total lipids. Seed oil is distinctive and this is attributed to the presence of high levels of omega-3-fatty acid at levels above any other natural oil 8,10 . Omega-3 fatty acids have many health promoting properties when consumed and is also an important raw material in cosmetic industry 10 . The aim of present study was to determine the variations in fatty acid compositions, tocopherol and sterol contents of several vegetable seed oils such as cabbage, radish, onion, lettuce, purslane, turnip, broccoli and carrot.

Material
The samples were collected by hand in August in 2018 from plants growing at several locations of Turkey. Seeds were transferred to laboratory in polypropylene bags under cool conditions. Seeds were obtained from fruits by handprocessing and stored in glass jars at 18 until analysis. Detailed information related to the samples are given in Table 1.

Oil content
The oil content was determined according to the method ISO 659:1998 ISO 1998 . About 2 g of the seeds were ground in a ball mill and extracted with petroleum ether analytical grade in a Twisselmann apparatus for 6 h. All solvent was removed by a rotary evaporator at 40 and 0.03 atm for about 10 min. The oil was dried under nitrogen and stored at 4 until use. Before analysis, oil samples were filtered.

Fatty acid composition
The fatty acid composition was determined following the ISO standard ISO 5509:2000 ISO 2000 . The fatty acid methyl esters were identified by comparing the retention time of the samples and appropriate fatty acids methyl esters standards. The fatty acid methyl ester was injected 1 μL in a Varian 5890 gas chromatograph with instrument fitted with flame-ionization detector FID and a capillary column, CP-Sil 88 100 m long, 0.25 mm ID, film thickness 0.2 μm . The temperature program was as follows: from 155 ; heated to 220 1.5 /min , 10 min isotherm; injector 250 , detector 250 ; carrier gas 36 cm/s hydrogen; split ratio 1:50; detector gas 30 mL/min hydrogen; 300 mL/ min air and 30 mL/min nitrogen; manual injection volume less than 1 μL 11 .

Tocopherols
In this study, HPLC coupled with fluorescence detector was used. 20 μL of a solution of 250 mg of oil in 25 mL of n-heptane HPLC grade was directly injected to a Diol phase HPLC column 25 cm 4.6 mm ID Merck, Darmstadt, Germany used with a flow rate of 1.3 mL/min. The mobile phase used was n-heptane/tert-butyl methyl ether 99 1, v/v HPLC grade 12 .

Sterols
The sterol composition of the oils was determined following ISO/FIDS 12228:1999 E ISO, 1999 . After 250 mg of oil was saponified with ethanolic potassium hydroxide by boiling under reflux, unsaponifiable matter was separated on an aluminium oxide column on which fatty acid anions were retained and sterols passed through. The sterol fraction was separated from unsaponifiable matter by thin-layer chromatograph Merck, Darmstadt, Germany , re-extracted from the TLC material, and afterward, the composition of the sterol fraction was determined by GLC HP 5890 using betulin as internal standard. The compounds were separated on an SE 54 CB Macherey-Nagel, Düren, Germany; 50 m long, 0.32 mm ID, 0.25 μm film thickness . Further parameters were as follows:

Statistical analysis
The means were compared by the use of one way variance analyses ANOVA , and the differences between the values were analyzed by Duncan multiple comparison test. Importance of the differences between the means was given according to p 0.05 importance levels 13 .

Results and Discussion
3.1 The oil contents and fatty acid compositions of seed oil samples The oil yield of seed samples are shown in Table 2, and their contents changed between 15.89 g/100 g purslane and 38.97 g/100 g black radish . The oil contents of red radish, turnip, lettuce yedikule and nuts radish seeds were found similar. The oil yield in the investigated fruit seeds ranged from 11.8 sea buckthorn to 28.5 watermelon 14 . Oil contents of Allium cepa seeds changed between 10.7 and 16.6 15 . Sipeniece et al. 16 Table 2. Generally, fatty acid compositions of seed oils changed depending on plant variety and species. Palmitic acid contents of oils ranged from 2.2 turnip to 15.0 purslane . The highest stearic acid was found in purslane oil 4.4 mL/100 mL , followed by curly lettuce 3.1 , cabbage Yalova 2.6 , nuts radish 1. 8 and black radish 1.7 mL/100 mL . While oleic acid contents of oil samples range from 12.1 mL/100 mL turnip to 69.8 purple carrot , linoleic acid contents of oils ranged from 8.9 black radish to 57.0 onion . The highest linolenic acid  19 . While quantitative values of oleic acids of cabbage Yalova and purple carrot oils are found similar compared to olive oil, linoleic amounts of onion, curly lettuce and lettuce yedikule seed oils were found similar to linoleic contents that of sunflower oil. In addition, erucic acid contents of red cabbage, white radish, red radish, turnip, nuts radish and black radish seed oils were found similar to rape seed oils while linolenic acid contents of purslane and white radish oils are determine similar to linolenic acid contents of soybean oil. But, generally fatty acid contents were found different from the values reported in literature and these variations can be attributed to differences in plant varieties.

The tocopherol contents of seed oil samples
The tocopherol profiles of some vegetable seed oils are given in Table 3. The γ-tocopherols are the most abundant form which has antioxidant properties and participates in the protection of all cell membranes in the body by fighting against free radicals 19 . While α-tocopherol contents of oil samples range from 2.01 mg/kg purple carrot to 903.01 Table 3 Tocopherol contents of seed oils mg/kg .    ***nondetected mg/kg onion , γ-tocopherol contents of vegetable seed oils changed between 1.14 mg/kg curly lettuce and 557.22 mg/ kg purslane . γ-Tocotrienol content of purple carrot was found too much higher than those of results of other studies oils. The highest P8 was found in red cabbage oil 21.42 mg/kg . In addition, δ-tocopherol contents of oil samples changed between 0.90 mg/kg purple carrot and 43.41 mg/kg onion . However, the highest δ-tocotrienol was found in red cabbage oil 159.85 mg/kg . It could be concluded that tocopherol contents of seed oils are significantly dependent on plant variety. It was observed statistically significant differences among tocopherol contents of oil samples depending on plant species p 0.05 . But, it was not observed statistically significant differences among β-tocopherol contents of red cabbage, curly lettuce, turnip, cabbage Yalova, lettuce yedikule oil samples. Total tocopherol contents of onion 127.04 mg/100 g were found lower than that of the seed oil of red onion Amposta 1666 mg/ kg 19 21 reported that turnip and radish seed oils contained 0.10 and 2.85 mg/100 g α-tocopherol, 2.13 and 0.48 γ-tocopherol, and 2.22 and 3.33 mg/100 g δ-tocopherol, respectively. Gornas 22 determined 51.40-88.70 mg/100 g α-tocopherol, 27.92-69.49 mg/100 g β-tocopherol, 16.71-35.16 mg/100 g γ-tocopherol, 7.01-26.59 mg/100 g δ-tocopherol in crab apple seed oils. The composition of tocopherols in the Japanese quince seed oil is dominated by the α-tocopherol 97 , while the β-tocopherol and γ-tocopherol constituted only minor level below 2 of each 23 . Sipeniece et al. 16 determined 102.11-119.74 mg/100 g α-tocopherol, 1.55-1.96 mg/100 g β-tocopherol, 105.37-124.27 γ-tocopherol in seed oils of three cvs. Rondo , Darius , and Rasa of Japanese quince harvested in 2015, 2016, 2017, and 2018 year. Moringa seed oil contained 5.05 α-tocopherol, 25.40 γ-tocopherol and 3.55 mg/kg δ-tocopherol 7 . β-tocopherol, γ-tocopherol and δ-tocopherol contents of seed oil of red onion Amposta were determined as 19.58 , 47.44 , and 4.85 , respectively 19 . The levels of tocopherols and tocotrienols in seed oils are comparable with grape, chia and caper seed based on oil content. Differences in tocopherol contents of seed oils are depend on plant species. Tocopherols were found to stabilise methyl esters by reducing the rate of peroxide formation while present. In addition, α-tocopherol was able to improve some established quality criteria, e.g. by inhibiting the formation of phytosterol oxidation products at high temperature in oils. Table 4 shows the sterol contents of oil samples. As seen in Table 4, these oils can be considered rich in campesterin, brassicasterin, sitosterin, and stigmasterin. While campesterin contents of seed oils change between 203.2 purple carrot and 2808.5 mg/kg cabbage Yalova , stosterin contents of oil samples varied from 981.5 curly lettuce to 4843.3 mg/kg purslane p 0.05 . In addition, while brassicasterin contents of samples change between 8.3 purple carrot and 894.5 red cabbage , δ5-avenasterin contents ranged from 45.9 curly lettuce to 971.3 mg/kg purslane . Also, δ7-stigmastenol contents of samples were found between 7.3 white radish and 773.9 mg/kg curly lettuce . Total sterol contents of seed oils changed between 2960.4 purple carrot and 9185.1 mg/kg purslane . GLC analysis of the seed oils shows that Purslane oil is characterized the presence of several sterols. It was not observed statistically significant differences among clerosterol contents of white radish, onion and nuts radish seed oil. In addition, it was not observed statistically significant differences among δ5-24-stigmastadienol contents of red cabbage, onion, red radish and turnip seed oils. Among the samples, total sterol contents of onion seed oil was found higher than that of result reported 96.122 by Aiboudi et al. 19 . The oil of Cucumeropsis edulis seeds contained 0.02 7-dehydrocholesterol, 4.14 lupeol, 0.020 lanosterol, 0.01 cholesterol, 13.05 stigmasterol and 82.60 β-stosterol 8 16 . According to previous studies, β-tocopherol does not occur in plant oils or is found in negligibly low quantities, ranging from 0 to 2.80 mg/100 g, depending on oil 25,26 . High amounts of linolenic acid are unsuitable for oil-food products due to its reversion of flavor associated with autoxidation 27,28 . The extraction technique and genotype effect on the oil yield and profile/concentration of fatty acids, tocopherols, sterols in oil obtained from the seeds. Plant genotype is considered to be the key factors determining the profile of fatty acids and the concentration of bioactive compounds in the oil extracted from the seeds 23 . In addition to the fatty acid composition, the composition of tocopherol is an important characteristic feature to de- ** The different letters in the same column show statistically significant differences (p＜0.05).

The sterol contents of seed oil samples
***nondetected scribe the identity of vegetable oils 27 29 . When the results were compared to with literature, different values were found. Variation in factors such as light, genetic factor, variety, harvest time, physico-chemical properties of the soils, climatic factors, and rainfall from one year to the next may be among the most important factors. A given plant grown in different geographical locations may have very different oil contents, fatty acid and tocopherol composition and sterol contents 5,17,19,30 .

Conclusion
Fatty acid, tocopherol and sterols had important constituents of oils studied. The oil contents of red radish, turnip, lettuce yedikule and nuts radish seeds were found similar. Generally, fatty acid compositions of seed oils changed depending on plant variety and species. In addition, erucic acid contents of red cabbage, white radish, red radish, turnip, nuts radish and black radish seed oils were found similar to rape seed oils while linolenic acid contents of purslane and white radish oils are determine similar to linolenic acid contents of soybean oil. The levels of tocopherols and tocotrienols in seed oils are comparable with grape, chia and caper seed based on oil content. These seed oils can be considered rich in campesterin, brassicasterin, sitosterin, and stigmasterin.