Changes in the sterol compositions of milk thistle oil ( Silybium marianum L . ) during seed maturation

In this study, the total lipid content and sterol compositions were determined during the development of milk thistle seeds. The oil content increased to a maximum value of 36±1.7% and then declined to reach a value of 30.5±0.9% at full maturity. The sterol content of milk thistle seeds was affected by the ripening degree of the seeds. At the early stages of seed maturation, Δ-stigmastenol was the most abundant sterol followed by β-sitosterol. However, at full maturity, β-sitosterol was the most predominant sterol (46.50±0.8%). As the seed developed, campesterol and stigmasterol amounts increased, while Δ-avenasterol content decreased. It can be concluded that milk thistle seed oil has a characteristic sterol pattern comparable to the ones elucidated for olive oil and corn oil. The extracted oil from milk thistle seeds is rich in phytosterols and could be used in food preparation and human nutrition.


INTRODUCTION
Phytosterols are minor components of vegetable oils and form a major proportion of the unsaponifiables (Azadmard-Damirchi et al., 2005).The individual sterols and their relative proportions can be used to determine the identity of the oil and to detect adulterations.It has been reported that conventional refining does not significantly affect sterol composition.Phytosterol contents in vegetables are known to vary due to different factors such as variety, season, extraction and other technological procedures (Li et al., 2007;Cercaci et al., 2007).
Furthermore, Phytosterols are known to lower serum low-density lipoprotein (LDL) cholesterol levels by reducing intestinal cholesterol absorption (Miettinenet et al., 1995).Clinical studies confirmed that phytosterols have hypocholesterolemy, anti-inflammatory and anti-carcinogenic effects (Awad et al., 2007;Ronco et al., 1999;Berges et al., 1995).Therefore, phytosterols have been added to several functional food products such as yoghurt, milk (Lagarda et al., 2006), and some vegetable oils (Ntanios, 2001).These types of products are now available on the market and have been scientifically proven to lower blood LDL cholesterol by around 10-15% as part of a healthy diet (Jones et al., 2000).
Milk thistle is an important medicinal crop in Europe and has recently become more significant in North America (Zheljazkov et al., 2006).The medicinal compounds of value are found in the seeds of the plant.In Tunisia, milk thistle is a common wild plant, which grows in many regions, and the people from some regions, particularly in the center areas, eat the seeds of this plant.Milk thistle seeds have been used for more than 2000 years to treat liver diseases (Karkanis et al., 2011).These seeds contain silymarin (Engelberth et al., 2008) and 25% (w/w) oil (Wallace et al., 2005).The oil has to be removed from the seeds prior to the extraction of silymarin.Therefore, it is a by-product of silymarin production.The oil extracted from these seeds can be used as a cure for many diseases including viral hepatitis and cirrhosis (Fadhil et al., 2012).
There are a very few data in the literature on the phyosterol composition of milk thistle seed oil.The sterol pattern of Silybium marianum has been determined in Egypt (EL-Mallah et al., 2003), Jordan (Dabbour et al., 2014) and Iran (Fathi-Achachlouei and Azadmard-Damirchi, 2009).The sterol composition can be affected by geographical growing area, difference in varieties and ripening degree of the fruits (Casas et al., 2004;Stefanoudaki et al., 2001;Harrabi et al., 2007).No work has been published on the sterol composition during the development of milk thistle seeds.The aim of this study was to monitor oil and sterol accumulation during seed maturation.The data obtained is important for evaluating the potential of milk thistle seeds to be exploited as a new source of oil for nutritional, industrial and pharmaceutical applications.

Plant materials
Milk thistle seeds were collected from plants growing wild in Tunisia (region of Sousse), during April and June, 2012.Seeds were picked according to external color; green seeds were chosen as immature stage, mahogany brown seeds as the intermediate stage and dark brown seeds as the last stage of maturity (mature stage).100 g of seeds were dried at 50 °C and then ground to fine powder in a grinder.

Oil Extraction
The oils were extracted using petroleum ether in a Soxhlet extractor for 4 h.The solvent was initially removed using a rotary evaporator at 40 °C.Oil samples were placed at ambient temperature (25-35 °C).

Saponifi cation
The unsaponifiable fraction was determined by saponifying 5 g of oil extracts with 50 mL ethanolic KOH 12% (w/v) and heating at 60 °C for 1.30 h.After cooling, 50 mL of H 2 O were added.The unsaponifiable matter was extracted four times with 50 mL of petroleum ether.The combined petroleum ether extract was washed with 50 mL of ethanol-water (1:1).The extracted ether was dried over anhydrous Na 2 SO 4 and evaporated to dryness using a rotary evaporator.The dry residue was dissolved in chloroform for TLC analysis.

Thin layer chromatography
The unsaponifiable matter was separated into sub-fractions on preparative silica gel thin-layer plates (silica gel 60G F254) using one-dimensional TLC with hexane-diethyl ether (6:4, v/v) as the mobile solvent.The unsaponifiable fraction diluted in chloroform was applied on the silica gel plates.After development, the plate was sprayed with 2,7-dichlorofluorescein and viewed under UV light.The band corresponding to sterols was scraped, extracted three times with chloroform-diethyl ether (1:1, v/v), filtered to remove the residual silica, dried in a rotary evaporator and stored at −10 °C.

Analysis of sterols by GC-MS
GC-MS analyses were performed using a capillary HP-5MS column (30 m×0.25 mm I.D., 0.25 μm film thickness; Agilent Technologies) with gas chromatography (Agilent Technologies 7820A) coupled directly to the mass detector (Agilent Technologies 5975 series MSD).Helium was used as carrier gas, with a constant flow rate of 1 ml/ min.The injector and detector temperatures were 230 °C.The oven temperature was programmed from 150 to 320 °C at 10 °C•min −1 from 150 to 250 °C and at 5 °C•min −1 from 250 to 320 °C.Electronimpact mass spectra were measured at acceleration energy of 70 eV.Manual injection of 1 μL of the sterol solution was performed in the split mode at a 10:1 split ratio.The phytosterol compounds were identified by comparing their relative retention times and mass spectra with those of the authentic standard.The peaks were also confirmed by comparison with the Wiley 275.L Mass Spectral Library.

Statistical analysis
A statistical analysis was performed by using the Proc ANOVA in SAS (Software version 8).Duncan's Multiple Range Test was used.For each oil sample, three determinations have been made.

Lipid content
During seed maturation the oil content increased to a maximum value of 35.8±1.3% and then declined to reach a value of 30.5±0.9% at full maturity.More oil was synthesized during the early stage of seed development.Malekzadeh et al. (2011) reported that the total oil content of milk thistle seeds decreased under drought stress.In mature seeds, oil was stored in the form of oil bodies (Voelker and Kinney, 2001).The extracted oil from milk thistle seed has been suggested as suitable as an edible oil (EL-Mallah et al., 2003).

Unsaponifi able fraction
The results obtained showed that the amount of total unsaponifiable matter decreased during seed maturation (Table 1).The greatest change occurred during the early stage of seed development.Thus, the highest level of unsaponifiable matter (3.8±1.2%) was detected in immature seeds.At full maturity, the unsaponifiable lipid content of the studied seeds was 1.9±0.2% of the total oil.The total amount of unsaponifiable matter in olive oils ranged from 1 to 2% of the total lipids (Alonso-Salces et al., 2009).Overall, the level of unsaponifiable matter ranged from 0.5 to 2.5% of the total lipids (Małecka, 2002).These minor lipids greatly influence the organoleptic quality and stability of the oil (Alonso-Salces et al., 2009).The effectiveness of unsaponifiable matter in retarding oil deterioration has been demonstrated by many researches (Mohamed and Awatif, 1998;Gopala Krishna et al., 2003).
The unsaponifiable fraction is made up of minor constituents (sterols, triterpene alcohols, aliphatic alcohols, hydrocarbons, etc.), which may vary both qualitatively and quantitatively depending on genetic factor, climatic conditions, extraction and refining procedures, as well as storage conditions (Canabate-Díaz et al., 2007).4-Desmethylsterols were isolated from the unsaponifiable matter and represented 55.2% of this fraction and about 1% of the total oil.This high proportion shows that milk thistle seed oil is one of the richest natural products in phytosterols versus other vegetable oils frequently used in the diet, such as olive oil (0.17%) (Weihrauch and Gardner, 1978).These results are useful for the consumer and also for the oil producer.Indeed, the unsaponifiable fraction of vegetable oils has applications in cosmetics and pharmacology due to its biological properties.

Sterols composition in immature seeds
Phytosterols are important due to their impact on health.Therefore, readily available food products have been engineered to be enriched in phytosterols and marketed to help lower serum cholesterol and reduce the risk cardiovascular disease.4-Desmethylsterols are the major components of phytosterol matter in most vegetable oils (Azadmard- Damirchi and Dutta, 2006).Eight 4-desmethylsterols were detected in the oil analyzed (Figure 1).The changes in sterol composition during the development of seeds were  summarized in Table 2.The sterol content of milk thistle seeds was affected by the ripening degree of the seeds.At the early stages of seed maturation, Δ 7 -stigmastenol was the most abundant sterol followed by β-sitosterol.As the seed developed, the level of Δ 7 -stigmastenol reduced from 52.84±1.5 to 27.81±0.5% of the total sterol content.This result could be explained by the high activities of Δ 7 -sterol Δ 5 -desaturase and Δ 7 -sterol reductase during the early stages of seed development.In fact these two enzymes are involved in the conversion of Δ 7 -sterols to Δ 5 -sterols (Benveniste, 2002).The Δ 5 -sterols are mainly accumulated in the plasma membrane, where they are believed to regulate the membrane fluidity (Grandmougin et al., 1989).The amounts of campesterol and stigmasterol increased gradually during seed maturation.However, the level of Δ 7 -avenasterol decreased as the seed developed.The results obtained show that the levels of Δ 7 -campesterol and Δ 5 -avenasterol were relatively constant.The amount of cholesterol was very low in immature seeds (<2%).
The rate of cholesterol accumulation was found to be greatest at the late stage of seed maturation.
The results obtained show that cholesterol represents 3.91±0.3% of the total 4-desmethyl sterol fraction.It was lower than that in the milk thistle cultivars grown in Iran (9.5%) (Fathi-Achachlouei and Azadmard-Damirchi, 2009) and in the cold-pressed seed oil of the cultivar grown in Jordan (15.14%) (Dabbour et al., 2014).Consequently, it could be suggested that the unrefined oils obtained by extraction with an organic solvent had a lower level of cholesterol than the cold-pressed oils.Cholesterol is the predominant sterol in animal fats and fish oils, but is very rare in vegetable oils.It is known that cholesterol occurs in the sterol fraction of many vegetable oils as a minor component and usually amounts to 1% of the total sterol content (Phillips et al., 2002), significantly lower than that detected in the milk thistle seed oil.It has been recognized that plant sterols could reduce plasma cholesterol levels in humans (Ostlund et al., 2002).The mechanism of cholesterol reduction in the presence of phytosterols is based on the blocked absorption of it in the digestive tract.Since the level of cholesterol is low and in the presence of excessive amounts of phytosterols, it can be expected that its absorption will be minimal and the positive effect of phytosterols will overcome it (Ostlund et al., 2002).Furthermore, cholesterol biosynthesis in higher plants has not been studied extensively and thus, uncertainties exist in the sequence of intermediates.The enzymatic approach to understanding and controlling the formation of the sterol structure was hampered by the low existence of sterol enzymes in higher plants.Therefore, milk thistle seeds which had a higher level of cholesterol as compared with the other seeds could be a good example for the study of the cholesterol biosynthetic pathway.

CONCLUSION
In summary, this study provides useful information on the sterol composition in immature milk thistle seeds.Milk thistle seeds are a rich source of phytosterols with a potential for beneficial therapeutic activities.Tunisian milk thistle oil had a very lower amount of cholesterol as compared with the Iranian and Jordanian cultivars.The results obtained can justify the important value of milk thistle seed oil as an attractive candidate for use in food preparation and human nutrition.

TABLE 1 .
Total oil and unsaponifiable matter contents of milk thistle seeds collected at three maturity stages *% of DW: % of dry weight.