CHROMATOGRAPHIC ANALYSIS OF SILYBUM MARIANUM ( L . ) GAERTN . FATTY OIL

The present paper describes biochemical (fatty oil) composition of Silybum marianum (L.) Gaertn. of Moldovan origin. The oil content of the seeds was approximately 25%. Linoleic acid (C18:2), an essential polyunsaturated fatty acid, is the most abundant (48.88%), followed by monounsaturated oleic acid (C18:1, 31.94%) and saturated palmitic acid (C16:0, 7.61%). Also, saturated stearic (C18:0, 4.31%), arachidic (C20:0, 2.63% and behenic acid (C22:0, 2.30%) were identified. The minor fatty acids are represented by saturated myristic (14:0, 0.09%) and margaric acid, (17:0, 0.07%), monounsaturated eicosenoic (C20:1, 0.99%), palmitoleic (C16:1, 0.07%) and erucic acid (C22:1, 0.08%). The RP-HPLC analysis of tocopherols composition showed the main components: α-tocopherol (23.45 mg/100g) and γ-tocopherol (5.60 mg/100g). Based on the obtained results, the extracted oil from milk thistle seeds is rich in essential fatty acids (about 50%) and tocopherols (29.09 mg/100g) and it can be used in food preparation.


Introduction
Silybum marianum (L.) Gaertn. is an important medicinal plant belonging to the Asteraceae Dumort family.Native of the Mediterranean region, it is cultivated now in many countries.In the Republic of Moldova, this species is cultured and does not grow in the spontaneous flora.
As a medicinal plant, milk thistle has been traditionally used for centuries to treat liver diseases and, presently, it is one of the most commonly used herbs worldwide.The active component of dried fruit extract of S. marianum is silymarin, an isomeric mixture of three flavonolignans: silybin, the main and most effective compound, silycristin and silydianin [1][2][3][4].
The plant seeds also contain a high amount of oil [17,18].Numerous studies have been conducted on these species, growing in different regions of the world, particularly on their fatty oil compounds [19,20].
The aim of this work is to reveal the biochemical composition of S. marianum oil, more exactly the content of fatty acids by means of gas-chromatography (GC) and the contents of tocopherols in the fatty oil using high-pressure liquid chromatography (HPLC).

Experimental
Seeds samples of milk thistle (S. marianum (L.) Gaertn.)plant variety "Panaceia", 1 st year of reproduction were collected from experimental fields of medicinal and aromatic collection, Botanical Garden (Institute) of Academy of Sciences of Moldova.The plants were cultivated at a density of 50 x 60 m 2 without soil fertilizer and seeds were collected manually at full maturity in the second decade of July.Their humidity was about 5.5%.

Fatty oil (FO) extraction
A sample of grounded seeds (0.2-0.5 mm, 300 g), was extracted at reflux in a Soxhlet type extractor using light petroleum ether (b.p. 40-60°C) during 3 h.After filtration, the solvent was removed at reduced pressure.The obtained fatty oil (75.8 g, 25.27%) was further used for measurements and chromatographic analyses.

Fatty acid methyl esters (FAMEs) preparation
The oil samples (200 mg) were dissolved in hexane (4 mL) in a conic tube and 200 µL of 2 M methanolic potassium hydroxide solution was added.After vigorous shaking in a vortex for 1 minute, the samples were neutralized with potassium hydrogen phosphate.The organic layer, which contains FAMEs, was filtered and 1 µL was injected into the gaschromatograph [21][22][23].

Gas chromatography (GC) analysis of fatty oil
Qualitative and quantitative analysis of fatty acid composition was performed on a Varian CP-3800 gas chromatograph equipped with a flame ionization detector (FID).A fused-silica SP-2560poly(biscyanopropylsiloxane) capillary column (100 m x 0.25 mm i.d.; film thickness 0.20 µm) from SUPELCO was used for separation and operated under the following conditions: oven temperature program: from 120°C up to 240°C at a rate of 4°C/min and then kept at 240°C for 30 min; injector and detector temperatures, 250 and 260°C, respectively; carrier gas, helium at a flow rate of 2 mL/min; split ratio, 1:100; nitrogen at a flow rate of 30 mL/min, hydrogen at a flow rate of 30 mL/min and air at a flow rate of 300 mL/min [24].Identification of fatty acids methyl esters was made with a standard mixture of 37 esters of fatty acids (FAME MIX 37) from SUPELCO.The results are expressed %(w/w) FA.

Sample preparation for tocopherols analysis
The oil samples (2.0 g) were dissolved in methanol (50 mL) and ascorbic acid (0.5 g) was added.After vigorous shaking, 50% aqueous potassium hydroxide solution (5 mL) was added and the resulted mixture was refluxed for 35 min under nitrogen.After saponification, the samples were let to cool down and diluted with water (55 mL).The extraction step was performed in a dark separation funnel with a mixture (50 mL) of petroleum ether:diethyl ether (80:20, v/v).After the separation of phases, the organic layer was transferred into another dark separation funnel and mother liquid was extracted additionally twice with the same mixture of solvents.The combined organic phase was washed with water (150 mL) to the neutral stage, evaporated to dryness under reduced pressure and re-dissolved in methanol (10 mL) for RP-HPLC analysis.

RP-HPLC quantification of tocopherols from milk thistle oil
Chromatographic separation of tocopherols was performed using a high performance liquid chromatography (HPLC) equipped with a quaternary pump, column oven, autosampler and diode array detector (DAD) (Agilent 1200 series, USA).The analytic column was an RP Ascentis C 18 (250 mm; 4.6 mm; 5 µm; Supelco Analytical) equipped with a guard column and thermostated at 30ºC.Tocopherols were separated isocratically within 16 min using a mobile phase containing MeOH:H 2 O (97:3, v/v), at a flow rate of 2 mL/min and detected at λ= 292 nm.The concentrations of tocopherols were calculated with a 5-point calibration curve with external standards.The standard concentrations ranged from 1.12 to 66.5 µg/mL.
For analysis, 10 µL samples were injected into the HPLC system.Tocopherols were identified by comparing their retention times against commercially available standards (Sigma-Aldrich) [21,25].The results were expressed as mg of α-, γ-tocopherols/100g oil.

Conclusions
The milk thistle fatty oil of Moldovan origin has a biochemical composition comparable with samples of other origins reported before and can be used for the same purposes.
This study revealed that the seeds of S. marianum are a rich source of ω-6 polyunsaturated fatty acids (PUFAs) (almost 50%) and α-tocopherol (23.45 mg/100g) which are very good antioxidants.The composition of extracted oil was similar to sunflower oil and might be used as cooking oil or salad dressing oil, alone or mixed with the other oils very rich in ω-3 PUFAs.References 1. Dixit, N.; Baboota, S.; Kohli, K.; Ahmad, S.; Ali, J.
Silymarin: a review of pharmacological aspects and bioavailability enhancement approaches.Indian

Table 2 The content of tocopherols in S. marianum L. fatty oil.
Figure 6.14α-Tocopherol and 15γ-tocopherol.