Antioxidant Potential and Stabilization Studies of Sunflower Oil Using Sorbaria tomentosa Extract and its Cu(II)/Zn(II) Chelates

FATIMA IZHAR, MUHAMMAD IMRAN*, TANVEER HUSSAIN BOKHARI, MARIYAM YOUSAF, SHABNUM JAVED, SUMMIYA REHMAN, SHOOMAILA LATIF, ZUNAIRA ZULFIQAR, LIVIU MITU* Institute of Chemistry, University of the Punjab, Lahore-54590, Pakistan Department of Chemistry, Government College University, Faisalabad, Pakistan Center for Undergraduate Studies, University of the Punjab, Lahore, Pakistan University of Pitesti, Department of Chemistry, 1 Targu din Vale Str., 110040, Pitesti, Romania

Antioxidant Potential of Pure Extract of ,,Sorbaria Tomentosa" and its Chelates Evaluation of Total Phenolic Contents (TPC) TPC of Sorbaria tomentosa extract was determined by the reported method of [17]. Absorbance of three sample solutions [extract, Cu(II) chelate, Zn(II) chelate] was measured on Spectrophotometer at 765 nm by using gallic acid solution as standard. The results were expressed as gallic acid equivalents (GAE) (g/100 mL) of Sorbaria tomentosa extract. All samples analyses were done in three replications.

Measurement of Total Flavonoid Contents
Total flavonoids in plant extract were determined by colorimetric method according to protocol reported by [18]. Absorbance of three sample solutions [extract, Cu(II) chelate, Zn(II) chelate] were determined at 510 nm. Results were expressed as total flavonoids of Sorbaria tomentosa extract in milligram of catechin/100 g of dry content weight. All the samples were analyzed in three replications.

DPPH Assay
The DPPH radical scavenging effect of Sorbaria tomentosa extract was determined by a reported assay [19]. Measurement of absorbance was carried out at 515 nm against ethanol (blank sample). Results were expressed by following formula: The antioxidant effect of Sorbaria tomentosa aliquot was determined in an aqueous emulsion of β-carotene and linoleic acid by method reported by [20]. Absorbance was measured for each sample instantly at 470 nm and then after 15, 30, 45 and 60 minutes at 50°C. For each extract duplicate analysis was done.
Ferric Reducing Antioxidant Power Assay FRAP method was performed according to the described assay of [21]. The reaction mixture contained 10 µL of Sorbaria tomentosa extract/chelates, distilled H2O (30 µL) and freshly prepared FRAP reagent (300 µL). Measurement of absorbance was done against each solution at about 593 nm. Results were expressed in terms of concentration of FeSO4.7H2O.

ABTS Radical Cation Inhibition Activity
This assay was performed by mixing 0.1 mL of plant extract/metal chelate/BHA/BHT and 0.9 mL of ABTS radical cation solution. The resulting mixture was kept for few seconds and then placed in dark for five minutes. Absorbance was determined at 734 nm against water : ethanol (50:50) as blank [22]. Results were calculated by the formula given below:  (20 mL). Then both the solutions were mixed in round bottom flask and stirred for about 3 to 6 hours at room temperature. Precipitates were formed, which were allowed to settle down over night and subsequently separated by centrifugation. Finally, the resulting precipitates were stored in dried vials for determination of their antioxidant potential [23]. FT-IR studies was conducted to check the formation of metal chelates.

Stabilization of Sunflower Oil Samples Preparation
Refined bleached deodorized (RBD) sunflower oil (SFO) was obtained from a local refinery in Lahore. Ratio of sample to oil was kept as 1:1. SFO was taken and 250, 500 and 1000 ppm of Sorbaria tomentosa ethanolic extract was added in it. Similarly, BHA and BHT (200 ppm) were also added separately in SFO for comparison. Control was prepared by adding only SFO without extract [24]. During the storage period of 45 days all samples were analyzed and should be kept air tight.

Estimation of Peroxide Value (PV), Free Fatty Acid Value (FFA) and Iodine Value (IV)
Peroxide value, free fatty acid value and iodine value of sunflower oil was investigated by using Sorbaria tomentosa ethanolic extract, synthetic antioxidants (BHA and BHT) at regular intervals of 15 days over storage period of 45 days by following the reported AOAC method [25]. Meanwhile, all parameters were also measured for control sample.

Statistical Analysis
All analysis were carried out in three replicates and results were expessed as mean ± standard deviations. Significant difference among all readings (p < 0.05) was calculated by applying one way analysis of variance (ANOVA) on statistical package for social sciences (SPSS).

Results and discussion
Percentage yield of the plant extraction in ethanol was determined and it was 9.9 ± 0.01 %. Phytochemical screening of Sorbaria tomentosa extract showed the presence of alkaloids, tannins, flavonoids, carbohydrates, saponins and phenols as major phytochemicals while cardiac glycosides and proteins were present in small amount (table 1). All these phytochemicals are bioactive compounds which are responsible for antioxidant activity, antimicrobial and antiinflammatory activities. Antioxidant potential is attributed to phenols and flavonoids [26]. FT-IR spectra of Sorbaria tomentosa and its Chelates FT-IR studies showed different stretching frequencies in pure extract and its chelates as shown in (Fig. 1). In Sorbaria tomentosa extract, different peaks were observed at 3350 cm -1 , 2928 cm -1 , 1618 cm -1 , 1401 cm -1 , 1041 cm -1 which showed the presence of ν -OH, ν -CH, ν -NH2, ν -CH2 and ν -C-O (ester) respectively. The various changes/shifts of stretching frequencies in Cu(II)/Zn(II) chelates (Fig. 1) than those observed in pure extract of Sorbaria tomentosa showed the involvement of various functional groups of extract in chelation with metal ions [27]. Phenols are main ingredients which imparts antioxidant potential to plants. Flavonoid is an important phytochemical found in plants which possess anti-allergic, anti-fungal and anti-inflammatory activities [28]. (Table 2) showed that the extract has greater amount of phenolic and flavonoids contents as compared to its chelated extract. Therefore, it is concluded that Sorbaria tomentosa extract contained maximum antioxidants compared to chelated extracts (S.T. > Zn-S.T. > Cu-S.T.) and can be utilized as a source of natural antioxidant easily.

FRAP Assay
This assay involves reduction of Fe +3 to Fe +2 and finally light blue colored solution is formed upon the addition of antioxidant containing substance [29]. (Table 2) showed that the trend of antioxidant potential was in the order of (S.T. > Cu-S.T. > Zn-S.T.). It reveals that S.T. possesses good antioxidant potential as compared to its chelated extracts (Cu-S.T., Zn-S.T.). DPPH Assay DPPH is a stable radical and is widely used for the determination of antioxidant potential of extracts [30]. The values of % inhibition were in the range BHT (65.70 − 74.84), BHA (65.67 − 72.49), S.T. (19.40 − 63.11), Cu-S.T. (14.28 − 44.35) and Zn-S.T. (12.58 − 40.29) and are graphically represented in (Fig. 2). This figure shows that % inhibition was increased by increasing the concentration (0.2-1 mg/L) of Sorbaria tomentosa extract and respective chelates. The observed order of % inhibition was as follow: BHT > BHA > S.T. > Zn-S.T. > Cu-S.T.; Fig. 2. Radical inhibiting activity of standard antioxidant (BHA and BHT), extract and chelated extract products β-Carotene Linoleic Acid Emulsion System β-Carotene linoleic acid is a useful method for the evaluation of antioxidant activity. It has been reported that the rate of β-carotene decolorization can be reduced in the presence of antioxidants [31]. (Fig. 3) shows the graph between absorbance versus time at 470 nm. It can be concluded from this graph that with the passage of time (0-60 min), Sorbaria tomentosa pure extract showed absorbance values (0.085-0.050), Cu(II) chelate (0.090-0.067) and Zn(II) chelate (0.088-0.058). This reveals that in case of Sorbaria tomentosa absorbance values decreased indicating reduction in β-carotene decolorization hence proving it better antioxidant than its chelated forms. However, it has lesser potential when compared with synthetic BHT and BHA having absorbance values in the range (0.090 -0.067) and (0.088 -0.058) respectively. ABTS method ABTS radical cation scavenging activity was also determined for the assessing the antioxidant potential S.T. extract and its respective chelates. The dark bluish green color of ABTS radical was decreased on addition of the extract and respective chelates. (Fig. 4) represents a graph between concentration and % inhibition (antioxidant activity). This figure depicts similar trend as outlined in above methods thus revealing following order: BHT > BHA > S.T. > Zn-S.T. > Cu-S.T.;  Triglycerides present in oils get hydrolyzed due to the contact with moisture, resulting in the formation of free fatty acids. More will be the formation of free fatty acids, more will be the rancidity of oil [35]. (Fig. 6) shows the increasing trend in FFA values for all samples (control, BHT, BHA, S.T.) with passage of time. Maximum increase was observed in SFO with control which was 2.56 ± 0.01 to 4.9 ± 0.00 (%) whereas minimum FFA value was shown by SFO with BHT (2.37 ± 0.01 % to 2.95 ± 0.01 %) from zero to 45 th day. SFO with BHA and S.T.(1000 ppm) exhibited FFA comparable values at 45 th day which was 3.40 ± 0.00 % and 3.41 ± 0.01 % respectively. The observed trend was: BHT < BHA = S.T. (1000 ppm) < S.T. (500 ppm) < S.T. (250 ppm) < Control, as illustrated in (Fig. 6).
These results are in agreement with our previous findings [23,33,34]. Iodine Value Iodine value denotes the degree of unsaturation of fatty acids (fats, oils or wax) and expresses the amount of iodine absorbed by fats or oils. More decrease in IV, more will be the rancidity of oil [36]. Iodine values of (control, BHT, BHA, S.T.) were measured and are graphically represented in (Fig. 7). Generally, it was observed that iodine value decreased with the passage of time in all samples. Least decrease in iodine value (gI2/100g) was observed in SFO-BHT while maximum decrease in IV was observed in control sample during the period of oil storage. From (Fig. 7), the decrease trend of IV can be adduced as follow: BHT < BHA = S.T. (1000 ppm) < S.T. (500 ppm) < S.T. (250 ppm) < Control.
These results are in agreement with our previous findings [23,33,34].

Conclusions
Phytochemical studies of Sorbaria tomentosa extract showed that it possesses phenols, flavonoids, alkaloids and many other phytochemicals which imparts antioxidant properties to it. Among Sorbaria tomentosa and its chelates, the pure extract of Sorbaria tomentosa exhibited more antioxidant potential. (S.T.) (1000 ppm) ethanolic extract which was used further for the stabilization of sunflower oil, proved its oil stabilization efficacy comparable with standard synthetic antioxidant like BHA.