EVALUATION THE ANTIOXIDANT ACTIVITY O F SESAME COAT AND SESAME CAKE EXTRACTS

This study was aimed to evaluate the anti-oxidant activity of mmthanol extracts from roasted and unroasted sesame seeds cake (RSC, URSC) and ethanol extracts from sesameccoat (SC) usingDDPPH and reducing power activity (RPA) assay. The quantitative and qualitative analysis of obtained extracts were done using RP-HPLC technique, the results revealed that the extracts were contained anti-oxidents(lignans ) sesamin ,ssesamolin andssesamol in different amounts Also the RPA of URSC extract at (10, 20, & 50 mg/ml) was similar to that ofbBHT , while that for RSC was similar to BHT at 30 mg/ml. Additionally, The RPA SC extract was comparable to that of BHT at ( 20 , 30 , and 50 mg/ml). It has been noticed that the radical scavengingaability (RSA) of BHT was greater than of the experimental samples that evaluated by DPPH assay. Mean time the RSA of the studied extracts was increased with increase of extracts concentrations from 10-50 mg/g. Based on the results of this study, sesame coat, sesame cake (from roasted and unroasted sesame seeds) extracts could be used asppotential natural anti-oxidant topprotectooil-rich food to avoid theppossible risks resulted from using the syntheticaantioxidants to prevent food.


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
antioxidants. Synthetic antioxidants have undesirable effects which lead to many health risks such as heart disease, carcinogenic effects and others (21). Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have limited use in food (5), while the use of tertiary butyl hydroquinone (TBHQ) is not permitted in many countries (15). Sesame seeds (Sesamum indicum L.) is one of the important sources for edible oil, it contain about448-55% oil. It is also rich in protein (220-25%), the residual of oil extraction process is known as sesame cake which contains around 500% protein depending on the extraction method. Sesameccccake extractsccccccontainvvvarious lignans (sesamin,ssesamolin andssesamol) that are phenolic compounds with antioxidant activity (14, 18) Sesame seeds coat contain phenolic components and other components which has antioxidant activity. The presence of natural antioxidants (γ-tocopherols, sesanin and sesamolin) in sesame seeds coat enhances the ant-oxidative stability of whole sesame seeds oil (1). Hence, many researchers focused on investigating for natural antioxidant to replace synthetic antioxidant, the natural antioxidant of plant origin (fflavonoids,ttannins, coumarins,ccurcumanoids,xxanthons, phenolics, lignans …etc) which presences inffruits,lleave,sseeds and oils are known topprotect lipids and lipid containing foods from oxidation (6). In recent years there is a greet tendency toward conversion of bio-waste to value added compounds, Shui and Leong (17) stated that the antioxidant components of agriculture industrial waste are not only useful for increasing food stability by preventing lipid per-oxidation but also protect biomolecules and supramolecular structures such as membranes and ribosomes from oxidative damage. Seed wastes from fruit processing and those derived from oil seeds are possess a variety of bioactive substance with many medical properties (12). Chang et al. (2) reported that the sesame coat ethanol extracts analysis using HPLC technique was showed the presence of the lignan (sesamin and sesamolin). Accordingly, the present study carried out to extract the antioxidant compounds from locally cultivated sesame seeds coat and sesame cake and evaluate their antioxidant activities using DPPH and reducing power assay.
Particle impuritiesssuchaasadust,ssands,sstones,sspoile d seeds,ssmall wweed seeds and othereeextrammmmaterials were separated by sieves, the sesame seedsccoat were removed andsstored at 4ºC until used. Sesame cake was obtained from roasted and unroasted sesame seeds by extracting the oil as described by Hadeel and Khalid (4).

Extraction andppurification of lignans fromssesame ccoat
The extraction method was adapted from Chang et al.
(2). Ten grams of sesame coat blended with 100 ml of ethanol using magnetic stirrer at room temperature for 12-16 hour. The ethanol extract was separated and the residual from thefffirst extraction was subjected to aaasecond extraction with a freshiimpulse of ethanol(ETH). The ethanol extracts were concentrated using rotary evaporator at 40ºC. The obtained extract was subjected to qualitative and quantitative analysis by RP-HPLC. Extraction and purification of lignans from sesame cake Fifty grams sesame cake from each roasted and unroasted sesame seeds (SCR, SCUR) were defatted with 400 ml n-hexane in soxhlet apparatus at 60ºC for 6 hour. Forty grams of defatted sesame cake was treated with 222 ml of NaCL 10% (w/v) and soaked for 1 hour withoooccasionally stirring. This process was repeated two more times at 1:3 (v/v) ratios. Then followed by washing with water forttthree times also at 1:3 ratios, the residue obtained after thesewwwashings was dried at550°C. Ten grams of the dried residue was extracted with methanol (200 ml) in soxhlet apparatus for 12 hour. The methanol extracts were concentrated using rotary evaporator at 60ºC, according to method described by Lieu and Dang (10). The final extract was identified by HPLC.

RP-HPLC analysis
Sesamol,ssesamin, andssesamolin content of sesame coat and sesame cake extracts were analyzed by (HPLC). Standard sesamin, sesamolin and sesamol) were dissolved in methanol (1mg/ml). Standards and sample were filtered through appolytetrafluoroethylene membrane filter (0.458 mm _ 13 mm; National Scientific Co. Lawrenceville,GA, USA). The filtrates were injected into a shimadzo HPLC system model LC-2010 A HT equippedwwith accC18 columnee(44.6 mm _1150 mm; id, 5 mm;wWaters Co.,mmmMilford,MA, USA). The mobile phase was a mixture ofmmmethanoll andwwwater (770:30,vv/v) at affflow rate of 0.5 ml/min, The injection volume 0.2 µl, a UVdddetector was set at22290 nm and column temperatures were set at 30C°, according to the method described by (9), extracts sesame coat and sesame cake and standard were injected into HPLC to determine their retention time.

Reducing Power
Therrreducing power(RP) of sesame coat and sesame cake extracts was determinedaaaccording to the method described by (13) with some modification.aaAliquot of111.5 ml of variouscconcentrations (10, 20, 30, 40, 50 mg/ml) from sesame coat and sesame cake extracts and BHT (as a control) were mixed separately with 1.5 milliliter phosphate buffer (0.2M, pH 6.6) and 1.5 milliliterpppotassium ferricyanide 10 mg/ml. Thewmixture wasiiincubated at 50°C/20min. then 1.5 milliliter of trichloroacetic acid (TCA) wasmmadded to themmixture, which was then centrifuged at9650 g for10 min. Equal volumes of supernatantio(1.5 ml) and distilled water (1.5ml) was mixed with ferric chloride (0.3 ml, 1.0%), and then the absorbanceingof the mixture was measured at85700 nmmmusing a spectrophotometer. Higher absorbance of themreactionumixtureiiiindicated greaterrreducing power. DPPH Free Radical Scavenging Assay DPPH assay was performed according to Kitts et al (8). One milliliter of 0.1mM DPPH solution was mixed with one milliliter of various concentrations (10, 20, 30, 40, 50 mg/ml) from ethanol extracts of sesame coat and methanol extracts of sesame cake and BHT (as a control) and vortexed thoroughly. The solutions were kept at room temperature (in dark) for330 min and absorbance was measured at 517 nm against a blank using spectrophotometer.The radical scavenging activities (%) were calculated according to the following equation: DPPH radical scavenging activity (%) = [(Abs control -Abs sample )/ Abs control ] χ 100 Abs control is the absorbance of DPPH radical Abs sample is the absorbance of DPPH radical + extracts from sesame coat and sesame cake and the standard antioxidant (BHT).

RESULTS AND DISCUSSION
The RP-HPLCmmethod was used for the quantification ofssesamol, sesamin andsssesamolin in sesame cake and sesame coat extracts. HPLC analysis of standard sesamol, sesamin and sesamolin wererrrun separately and gave single peak with retention times (RTs) of 4.08, 4.41 and 2.51 min respectively (Fig.1). Figure (2) illustrates the RP-HPLC profile of sesame coat extract in ethanol, sesamol, sesamin and sesamolin were appeared at 2.68 (peak 1), 3.98 (peak 3) and 4.34 (peak 4) min, respectively, the amount of the above mentioned components were 120.46, 235.82 and 175.29 mg /100g sesame coat . Identification of sesamol, sesamin and sesamolin based on comparing their RTs with those of the standard compounds (Fig.1), obviously, sesamol, sesamin and sesamolin are proven to present in sesame coat extracts. These results agree with Chang et al.,(2) who found sesamin, sesamolin and very small amounts of sesamol in the sesame coats extract, using RP-HPLC analysis. He also mentioned that ethanol is safer than methanol and acetone from thetttoxicological point of view. Additionally, using ethanol as a solvent reduces the sesame coat oxalic acid content; therefore it decreases the risk of Ca-oxalate kidney stone (11)

Figure 2. RP-HPLC Chromatograms of sesame coat extracts in ethanol
According to Fig.(3) RP-HPLC analysis of the extracts of sesame cake from roasted and unroasted sesame seeds, sesamol, sesamin and sesamolin were appeared at 2.60 (peak 2), 3.93 (peak 3) and 4.48 (peak 4) min, respectively, sesame cake from roasted sesame seeds contained 642.61, 199.55 and 68.96 mg/100g sesame cake of sesamol, sesamin and sesamolin, respectively, while sesame cake from unroasted sesame seeds contained 707.81, 441.80 and 212.94 mg/ 100 g sesame cake of sesamol, sesamin and sesamolin, respectively. Therefore, sesamol, sesamin and sesamolin are proven to present in extracts of sesame cake from roasted and unroasted sesame seeds These results is in agreement with Suja et al.,(18) analysis. Shamurad, (16) found that oil from roasted sesame seeds contained higher amount of sesamol as compared to oil from unroasted seeds. The latter coumpound is known to be a strong antioxidant agent (7) Figure 3. RP-HPLC Chromatograms of sesame cake from unroasted sesame seeds (A) and sesame cake from roasted sesame seeds(B Table (1) shows the anti-oxidative-ability of sesame coat and sesame cake from roasted and unroasted sesame seeds extracts and compared to that of BHT. The reducing ability of all sample under study showed a concentrations dependent increases, as the concentration increased the reducing ability increased. It has been noticed that the reducing power activity of sesame coat and RSC at all concentrations (10, 20, 30 40, 50 mg/ml) were significantly lower than BHT, while there were no significant difference among reducing power activities of URSC and BHT at all concentration except at (30, 40 mg /ml), where URSC was significantly higher at (30 mg/ml) and the BHT were significantly higher (at 40 mg/ml) in reducing power activities. URSC reducing power activities were significantly greater than RSC and sesame coat at (30, 40 and 50) mg/ml. The superiority of URSC in reducing power could be attributed to the presence of sesamol in higher amount as compared to the RSC and seeds coat extracts. Chang et al.,(2) reported that the reducing power of the sesame coats extractiiiiiiiincreased withiiincreasing amount of extract, these results were agreed with Xie et al. (20) who reported that the extract concentrations an important factor in enhancing anti-oxidant activity. The antioxidant functionality mechanism of sesame coat extract could be due to its ability as scavenger of hydroxyl radical besides its ability in donating-hydrogen. Esmaeilzadeh Kenari et al.,(3) found that the reducing power of sesame cake extract in methanol and in ethanol were lower than BHT  among their radicals scavenging activities at (20,30 and 40 mg/ml), at 50 mg/ml the seeds coat extract RSA was significantly greater than that of RSC. In this regard, Chang et al., (2) found that the radicals scavenging activities(RSA) of sesame coats extract(SCE) was less than BHT. Esmaeilzadeh Kenari et al., (3) found the scavenging effect of sesame cake methanol extract on DPPH radical was less than BHT. Suja et al. (18) stated that anti-oxidant activity of sesame cake extract was concentration-dependent, and the purified extract was more effective than BHT, while the crude extract activity was comparable to to that of BHT. Moreover, Suja et al. (19) proposed that SCE could be a good alternative for synthetic anti-oxidant to protect the vegetable oils which contain different levels of unsaturated fatty acids.