The lipid-soluble vitamins contents of some Vicia L . species by using HPLC

In the present study, lipid-soluble vitamin compositions in the seeds of the Vicia L. taxa (V. ervilia (L.) Willd., V. cuspidata Boiss., V. peregrina L., V. cracca L. subsp. stenophylla Gaudin, V. mollis Boiss.& Hausskn., V. hybrida L., V. sativa L. subsp. nigra (L.) Ehrh. var. nigra L., V. sativa L. subsp. sativa (Ser.) Gaudin var. sativa, V. crocea (Desf.) B. Fedstch., V. noeona Reuter ex Boiss. var. noeona, V. narbonensis L. var. narbonensis) were determined by using HPLC. It was found that studied Vicia species apart from V. ervilia and V. cuspidata have highest ß-carotene contents, 1523,7±6,4 μg/g and 236,62±1,8 μg/g, respectively. Also, this study showed that V. ervilia and V. cuspidata have highest γ-tocopherol content. On the other hand, current study indicated that Vicia species have D3 vitamin contents between 13,8±0,62 μg/g and 50,5±2,13 μg/g. However, the αtocopherol, α-tocopherol acetate, D2, K1, retinol and retinol acetate contents of studied Vicia L. species were lowest. ARTICLE HISTORY Received: January 08, 2019 Revised: February 22, 2019 Accepted: March 14, 2019


INTRODUCTION
Fabaceae, is the third major family of higher plants, contains about 730 genera and 19400 species and are second to Poaceae in agricultural and economic priority [1][2][3][4].The members of family are harvested as yield and used in many areas such as medicines, fuel and chemistry [5].They are oldest crops consumed as important source of protein in the various areas of world including Europe, Middle East, Africa, and South Asia [6][7][8].These are rich in carbohydrates, proteins, minerals, polyunsaturated fatty acids, fibers, carotenoids and vitamins [9][10][11].
Not only plants consumed as food they can also used as the therapeutic effects against different diseases [26,27].Although some studies performed about determining phenolics, proteins and fatty acid compositions of legumes including Vicia there were no enough information about the lipid-soluble vitamins contents [28][29][30][31].Vitamins are important both human metabolism and also redox reactions in the plants [32].Lipid-soluble vitamins including tocopherols and carotenoids, are non-enzymatic components of antioxidant system, have important role to protect cell membranes against free radical damage by working together [33][34][35].Vitamin E, is a most powerful interrupting chain breaking antioxidant, defends cell membrane fatty acids agaist lipid peroxidation [36].Also, carotenoids supply antioxidative preservation against lipid rich tissues [37][38].It has been demonstrated that there is an inverse linking between the dietary intake of antioxidant-rich food and apparance of human diseases [39].Studies showed that α-tocopherol, γ-tocopherol, ß-carotene, lycopene and lutein inhibit cancer, coronary heart disease, neurological diseases, diabetes [40].
The major goal of present study is to determine the lipid-soluble vitamin contents of some Vicia L. taxa.Hereby, it was intended to contribute the biochemical studies of legumes which not enough study about vitamin contents.It is thought that such biochemical studies will provide support to researchers working legumes and also antioxidants.

Collection of Plant Materials
Sample plants were gathered from the natural habitats and were stored in the Firat University Herbarium (FUH;4001-4011).The species were identified by Dr. Ahmet Şahin and Dr. Hasan Genç.Details about the materials are explained in Table 1.

2.2.Extraction of plant materials
1 g seed used to analyse the lipid-soluble vitamins contents.The seeds are homogenised with hexane/isopropanol (3:2 v/v) [41].The solvent was removed on a rotary evaporator at 40°C after samples were centrifuged at 10.000 rpm for 5 minutes.After that lipid-soluble vitamins were extracted according to the method of Sánchez-Machado [42].Laboratory works were repeated three times.

Chromatographic analysis and quantification of lipid-soluble vitamins
Samples were vortexed and kept at 85 ºC for 15 min.After 5% KOH added to samples to determine the vitamin contents.Following, the tubes were cooled to room temperature and pure water was added and stirred.The non-soaped lipophilic molecules were extracted with 2x5 ml hexane.Later, hexane phase was evaporated using nitrogen gas.Finally, residue was solved 1 ml methanol/acetonitrile (25/75 v/v) and taken to autosampler tubes.
The column used was a HPLC Column A Supelcosil TM LC18 column (250 x 4.6 mm, 5 mm, Sigma, USA).The elution was performed at the flow rate of 1 ml/min.The analytical column was worked at 40 °C.One ml extract gained by saponification and it is subtilized by using 500 µl of the mobile phase.It was used LC-10 ADVP UV-visible as pump, SPD-10AVP as detector, CTO-10ASVP as column furnace, SIL-10ADVP as otosampler, DGU-14A as degasser unite.Detection was conducted at 320 nm for retinol acetate, retinol, 215 nm for vitamin D2, D3, γ-tocopherol, α-tocopherol acetate, α-tocopherol, 235 nm for vitamin K1 and 450 nm for ß-carotene.Identification of the vitamins were performed by external standard mixture.Class Vp 6.1 software assisted at workup of the data [43].The results of analysis were uttered as μg/g for samples.

CONCLUSION
In conclusion, this study presented that studied Vicia taxa have highest β-carotene contents except for V. ervilia and V. cuspidata which have highest γ-tocopherol content.Also, studied Vicia taxa have high D3 vitamins.It was fouınd that the other studied lipid-soluble vitamins low amounts or absent in this study.

Conflicts of Interest
Authors declare no conflict of interest.

Table 1 .
Localities of studied Vicia L. taxa

Table 2 .
The lipid-soluble vitamins contents of studied Vicia L. taxa