CHARACTERIZATION OF WHITE SESAME SEED OIL AND ITS BIOACTIVE COMPONENTS

125 ± 0.01), polyphenol mg/kg (Gallic acid 0.985 ± 0.001, protocatechuic acid 0.018 ± 0.001, syringic acid 0.024 ± 0.002, ferulic acid 0.134 ± 0.001), stigmasterol 6689.34 ± 0.02 and lignans mg/kg (sesamin 18340 ± 8.5, sesamolin 28340 ± 13.7 and sesamol 224 ± 2.0) were quantified. The results show that WSSO from variety PB-Till 90 has good physicochemical properties and contain appreciable quantities of bioactive components to make its usage as a functional food ingredient.


INTRODUCTION
As a result of research interventions, the concept of nutraceutical and functional foods has flourished.Now, consumers have started to look at food not only for its basic nutrients but also for its health benefits.Now it's the responsibility of nutritionists and researchers to emphasize their concentration towards health claims and safety issues of new foods.The major emphasis of these foods is on recuperating health and minimizing disease threat mainly through prevention.White sesame (Sesamum indicum) has its origin in India and roots in the history from more than 5000 years ago, belongs to the family Pedaliaceae and is considered as among one of the oldest crops on the planet (Bisht et al., 1998).Sesame has given the name of "queen" of oil seed crops because of the superb quality of the consumable oil it produces (Akinoso et al., 2010).It serves as a healthy food for humans because of its elevated level of superior quality oil that ranges between 42-54% (Orruño and Morgan, 2007).Sesame seed flour contains carbohydrates (5-9 %), protein (19-25 %), fiber (3-5 %) and some minerals such as calcium, phosphorous in abundance and vitamin E (Weiss, 2000).Sesame oil is a good source of unsaturated fatty acids which are approximately 80% with 37.2% of the oleic and 46.3 % of the linoleic acids (Weiss, 2000).Sesame seed contains very high levels (up to 2.5%) of furofuran lignans (mainly Sesamin, sesamolin, and sesaminol glucosides) with beneficial physiological activities (Uzun et al., 2008).Bioactive components in sesame seed oil show positive effects against chronic diseases like blood pressure, lipid profile and hyperglycemia (Jannat et al., 2010).Sesame seed lignans (Sesamin and sesamolin) reported to have various health benefits including modulation of fatty acid metabolism, cholesterol lowering effect through inhibition in absorption and biosynthesis, antioxidant and vitamin E sparing effects, hypotensive effects and anti-aging effects (Hirata et al., 1996).Both these lignans show significant results against hypertension in humans by lowering the intestinal absorption of cholesterol, this effect is mainly due to sesamin that is enhanced by αtocopherol (Ogawa et al., 1995).Its consumption significantly increased the level of vitamin E in animals and helps to reduce the oxidative stress (Kamal-Eldin et al., 1995; Yamashita et al., 1992).Diabetes, migraines and hepatitis like chronic diseases have been treated with sesame seed oil in the recent years (Anilkumar et al., 2010).The lower quantity of saturated fat and higher quantity of monounsaturated and polyunsaturated fatty acids (PUFA) makes the white sesame seed oil (WSSO) suitable for daily consumption.On the other hand, sesamin and sesamolin are the major phenolic lignans found in sesame oil ranges from 1.0-2.0%(Reshma et al., 2010) and these have proven beneficial effects as they serve as an antioxidant, antihypertensive, antimutagenic, anti-inflammatory and antithrombotic and cardioprotective effects (Kaneez et al., 2007).White sesame variety PB Till-90 is one of the important and majorly grown sesame variety in Pakistan and has not been characterized especially for its nutritional value and other health benefits in detail.It is the reason that sesame oil utilization is not much in our routine life.Sesame seed oil is among one of the few vegetable oils that can directly be used without refining.The stability of sesame seed oil is due to the presence of bioactive components in it like sesamin, sesamolin, sesamol and tocopherols (Chang et al., 2002).Its healthy aspects make it suitable to be used in different products and as a whole natural salad oil.The purpose of the study was to evaluate the WSSO for physico-chemical properties, fatty acid profile and bioactive components like tocopherol, polyphenol, phytosterol and lignans.

Scientific hypothesis
The scientific hypothesis of this study is mainly determined the chemical profiling, fatty acid composition of extracted white sesame oil along with their antioxidant potential.Further, by conducting these analyses which is used to enhance the biological value and also used to prepare the different quality products through white sesame oil.

Materials, chemicals, standards and reagents
White sesame was procured from Ayub Agriculture Research Institute, Faisalabad, Pakistan.All chemicals, reagents, solvents and standards for bioactive components were of highest purity for laboratory analysis and were purchased from Sigma Aldrich-UK.

WSSO Extraction and Characterization
The WSSO was extracted from PB Till-90 seeds by solvent extraction method (Latif and Anwar, 2011) using n-hexane as organic solvent.After extraction by Soxhlet apparatus, oil was tested for various physical and chemical attributes including color, refractive index, specific gravity, smoke point and chemical characteristics like free fatty acids, peroxide value, thiobarbituric acid (TBA) acid value, iodine value, saponification value, unsaponifiable material and free radical scavenging activity by following the procedures described in AOAC.(2003).

DPPH free radical scavenging activity
Free radical scavenging activity was measured using 1,1-diphenyl-2picrylhydrazyl (DPPH), by adopting the method described by Bozin, Mimica-Dukic, Simin, and Anackov (2006).The absorbance was measured at 515 nm against blank.The percentage of radical scavenging activity (RSA) was calculated using the following equation: A0 is the absorbance of control and As is the absorbance of the sample at 515 nm.

Fatty Acid Profile
Fatty acid methyl esters (FAMEs) of sesame seed oil were prepared using the standard IUPAC method (IUPAC, 1987) as described by Latif and Anwar (2011).Fatty acid methyl esters were then analyzed against standards (Sigma Aldrich-UK) by gas chromatograph (model: 14-A, Shimadzu, Japan) using polar capillary column (SP-2330, 30m x 0.32mm) coated with methyl lignose, flame ionization detector and nitrogen gas as a carrier.

Tocopherols
The tocopherols (α, γ, and δ) were determined in sesame seed oil using High Performance Liquid Chromatography on UV detector at 295 nm following method explained by Latif and Anwar (2011).

Phytosterol
Sample preparation and analysis of phytosterols were performed by adopting the method of Ryan, Galvin, Connor, Maguire, and O'Brien (2006) with some modifications.The sample was loaded onto a column Luna C8 (2) (250 x 4.6 mm) Phenomenex, Cheshire, UK.All determinations were done at 205 nm for phytosterols.

Lignans (Sesamin & Sesamolin)
Lignans (sesamin and sesamolin) were analyzed by adopting the method of Schwertner and Rios (2012) with little modifications.The sample was injected into the HPLC (Model: Perkin Elmer series 200 USA) equipped with C18 (4.6mm x 150 mm).The UV detector was set at 288 nm.Sesamin, sesamolin and sesamol were quantified by comparing with standards Sigma Aldrich-UK.

RESULTS AND DISCUSSION
The physicochemical properties of crude WSSO are presented in Table 1.Colour of oil ranges from clear yellowish to slightly red, with results of 18.4 ± 0.2 yellow and 1.53 ± 0.153 red.Refractive index gives us an idea about rancidity and adulteration, the refractive index of sesame seed oil was (1.4598 ± 0.1), previously in an Indian variety it was reported 1.469 by Alyemeni et al. ( 2011), specific gravity is the ratio of density of a substance to the reference substance; water was the reference substance to measure the specific gravity.Sesame seed oil specific gravity was (0.9210 ± 0.0), the point at which the oil starts to burn is the smoke point, white sesame seed oil smoke point was 184 ± 3 ºC.The free fatty acid is the % age of oleic acid in the oil that indicated the oxidation level of lipids, it is directly related to acidic composition of oil, sesame seed oil free fatty acid value was (0.95 ± 0.05 %).The measurement of resistance against oxidation is the peroxide value, WSSO peroxide value was (0.88 ± 0.03 meq/kg) while thiobarbituric acid value is an important indicator of the oil quality because it tells about the rancidity of oil, sesame seed oil thiobarbituric acid value was (0.045 ± 0.0 mg malonaldehyde/kgoil).The iodine value is the measurement of degree of unsaturation of fatty acids, higher value of iodine value shows that it is a semi dried oil and unsuitable to be used in paint industry before dehydration (Fernando and Akujobi, 1987).Iodine value of WSSO was (113.4 ± 1.46 g/100g), in one of the studies it was 107 g of I/100gm, free radical scavenging activity (DPPH) is the measurement of inhibition of linoleic acid peroxidation, sesame seed oil (PB Till-90) showed DPPH activity as 85.07%, previously in one of the studies it was 62.2 μg/ml, saponification value is the measurement of chain length of fatty acids presents in it, higher proportion of it make it unsuitable to be used in soap industry, sesame seed oil saponification value was 189.7 ± 3.97 mg KoH/g, in one of the studies it was 169 mg of KOH/g of oil reported by (Latif and Anwar, 2011), unsaponifiable matter is the components in oil that fail to form soap, in our study it was 1.44 ± 0.03 %.

Bioactive Components
Results for bioactive components in sesame variety PB-Till 90 are presented in Table 3 Tocopherol contents in sesame seed oil were (α 8.12 mg/kg, δ 23.98 ± 0.02 mg/kg, γ 3.125 ± 0.01 mg/kg), while sesamin and sesamolin were the major fat soluble lignans, sesamin 18340 mg/kg, sesamolin 28340 mg/kg and sesamol 224 mg/kg (Table 3).The most important oil sources for lignans are flaxseed and sesame seed oils (Peterson et al., 2010;Reshma et al., 2010) and it is evident from several studies that they play very important role in management of cardiovascular diseases (Peterson et al., 2010) 2007) and they detected stigmasterol content in sesame oil as 41.5 mg/100gm.Lignans (sesamin, sesamolin and sesamol) are the oxidative coupling product of βhydroxyphenylpropane (Fukuda et al., 1986).These compounds increase the oxidative stability of the oil.Sesame seed oil has sufficient quantities of fat soluble lignans (Schwertner & Rios, 2012).It is cleared from the results that WSSO PB-Till 90 characterization and especially the presence of sufficient quantities of components like tocopherol, polyphenol, phytosterol and lignans make sesame seed oil distinguished from other vegetable oils and make it suitable for human consumption.

Strengths
No other study for the characterization that includes physicochemical properties, fatty acid profile and evaluation of bioactive components of white sesame seed oil PB Till-90 has conducted.GC/MS and HPLC techniques were used for the evaluation of fatty acid profile and bioactive components, respectively.

Conclusion and Future Directions
Physicochemical properties and bioactive components of sesame seed oil are unique.The results of characterization and the presence of bioactive components make it suitable for human consumption.The presence of MUFA and PUFA makes it a good replacement for other vegetable oils.All these properties make it suitable not only for human consumption but also to be used in food industry, the pharmaceutical industry and in cosmetics preparation.Comparison with other varieties of sesame seeds and other vegetable oils will give good comparison to highlight its different aspects.

Table 1
Physicochemical properties of sesame seed oil PB Till-90

.
It is the reason that several efforts have been done to improve the extraction process of lignans from edible oil sources(Latif and

Anwar, 2011; Reshma et al., 2010).
The present sesame variety i.e.PB Till-90 is an excellent source of sesamin and sesamolin.Higher contents of tocopherol in oil increase the oxidative stability of the oil.Latif and Anwar (2011) reported tocopherol levels as α 2.4 mg/kg, γ 578 mg/kg, δ 3.7 mg/kg using solvent extraction method and it can be improved using enzyme-assisted extraction.PB Till-90 polyphenol contents were Gallic acid 0.985 ± 0.001 mg/kg, protocatechuic acid 0.018 ± 0.001 mg/kg, syringic acid 0.024 ± 0.002 mg/kg and ferulic acid 0.134 ± 0.001 mg/kg.The presence of polyphenols was supported by the studies of Borchani et al. (2010), they reported polyphenols as 14.21 mg cafeic acid kg-1 of the raw sesame oil.Presence of polyphenols also enhance the antioxidant activity.

Table 2
Fatty acid profile of sesame seed oil PB Till-90

Table 3
Bioactive Components in sesame seed oil PB Till-90 Phytosterols, primarily β-sitosterol, campesterol and stigmasterol, are membrane constituents of plants that effectively reduce serum LDL cholesterol and atherosclerotic risk (De

Jong et al., 2003).
In our study stigmasterol was detected in the sesame seed oil that was 6689.34 ± 0.02 mg/kg.The presence of phytosterol was also supported by