Antioxidant Activity of Methoxylated Flavonoids in Oils in Deep Frying Processes

Methoxylated flavonoids isolated from cold acetone leaf wash of Polygonum senegalense; 5-hydroxy-7-methoxyflavanone 1 and 5-hydroxy-6,7-dimethoxyflavanone 3, were tested for their ability to enhance thermal stability of vegetable oils. Determination of the peroxide value (P.V.) and the p-Anisidine value (p-A.V.) was done according to the standard methods of analysis. The compounds were tested for in vitro cytotoxicity against a mammalian cell-line, Chinese Hamster Ovarian (CHO) using the 3-(4,5-dimethylthiazole2-yl)-2,5-diphenyltetrazoliumbromide (MTT)-assay. Studies on changes in peroxide and p-Anisidine values for the oils heated to temperatures between 180 ̊C and 200 ̊C recorded better stability enhancement at 100 ppm concentration with these flavonoids than the commercial antioxidant, butylated hydroxytoluene (BHT). The plant-based flavonoids had no significant cytotoxic effect against the CHO cell-line and may serve as alternative antioxidants to synthetic ones which have previously raised great concern over the health of consumers.


Introduction
Stability enhancement of fish oil is necessary to improve its shelf-life because fish oil contains highly unsaturated oils which are more susceptible to oxidation [1].The rate and extent of formation of oxidation products in oils depends on the nature of the fatty acid composition (monounsaturated or polyunsaturated), the temperature at which the oil is heated [2] and the presence of metal ions [3] O. M. Wanjau et al.DOI: 10.4236/fns.2018.9110911274 Food and Nutrition Sciences [4]).Fish oil largely comprises of arachidonic acid, C20:4ω-6, docosapentaenoic acid, C22:5ω-3, and docosahexaenoic acid, C22:6ω-3 [5] and is more easily oxidized than vegetable oil [6].Lipid oxidation is a highly deteriorative process and health disorders such as atherosclerosis and cancerogenesis among others correlate highly to the consumption of highly oxidized oils [7].
Edible fats/oils may contain up to 200 ppm of synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) [8], purposely added to improve the shelf life of the edible fats/oils.The use of synthetic antioxidants is, however limited, because of their physical characteristics and the unfolding toxicological concerns on some of the synthetic antioxidants [9].
Flavonoid aglycones are reported to be deposited on the surface of leaves, twigs and seeds of Polygonum senegalense and Psiadia punctulata, with a higher deposition on younger plant parts than on the older parts [10] [11]).The existence of an exudate on the leaf surface, rich in methoxylated flavonoids [12] [13]), is fascinating.The constituent compounds of leaf surface exudates [14] supposedly serve some protective role on the plant [15]).Epicuticular layer and external flavonoids not only reflect radiation, but are also known to be good quenchers of singlet oxygen [16].Some of the constituent compounds of the leaf surface exudates are relatively non-polar and could perhaps be endowed with invaluable bioactivity in the relatively non-polar fish oil and edible oils.The choice for an oil stability enhancer must critically address the solubility and thermal stability of the oil additive and evaluate their cytotoxicity levels.
The presence of some synthetic antioxidants in fats and oils may not guarantee stability at deep frying temperatures.Antioxidants of low boiling point may gradually vaporize and thus expose the oil to oxidation [17].The oil may degenerate into various oxidation products, some of which may have injurious effects to the body.Antioxidant principles, stable at high temperatures, must be identified for use as oil additives to fortify the heat stability of the oils.The concentration levels of antioxidants may continue to diminish through vaporization at the high temperatures, leaving the oil exposed to further oxidation [18].

Plant Materials
The resinous leafy branches of P. senegalense were harvested, along the river banks of Njoro river within Egerton University, Nakuru County, Kenya, at an altitude of 2300 m, twenty five kilometers, west of Nakuru town.A voucher specimen is deposited in the National Museum of Kenya Herbarium.

Extraction
One kilogram of leaves detached from the branches of P. senegalense plant were stuffed into a 5 L erlmeyer flask for extraction.A 1.5 liter portion of acetone was introduced into the flask and shaken for two minutes and decanted as an orange solution [19] two more fresh portions of acetone were used to wash the leaves

Determination of Peroxide and p-Anisidine Values
Sunflower and rapeseed oils and fractionated palm shortening (Rina oil and Chipsy fat and Canola oil) were procured locally from local market in Nakuru.
Two aluminium based cooking pots (15 cm in diameter each) were used for electrically heating the oil in pot A and B (control) respectively.250 g of each oil sample was electrically heated in the cooking pots up to a temperature of 180˚C -200˚C for 7 hrs each day.50 mg of the plant isolates were dissolved in 1 ml of acetone in a vial bottle and introduced into the oil in pot A.
The vial bottle was rinsed twice with 1 ml portions of acetone and the washings transferred into the oil.The second pot served as a control into which 3 ml of acetone was added into the oil.Determination of the peroxide value (P.V.) and the p-Anisidine value (p-A.V.) was done according to the standard methods of analysis [21].The experiment was repeated three times for each case.

Cytotoxicity Screening Tests
Compounds were tested for in vitro cytotoxicity against a mammalian cell-line, Chinese Hamster Ovarian (CHO) using the 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazoliumbromide (MTT)-assay [22].All samples were tested in triplicate on a single occasion.The MTT-assay is used as a calorimetric assay for cellular growth and survival, and compares well with other available assays [23] [24]).The tetrazolium salt MTT was used to measure all growth and chemosensitivity.Flavanone 1 (Polsen 1) was dissolved in 10% methanol while flavanone 3 (polsen 3) was dissolved in 10% DMSO.Compounds were tested as a suspension due to poor solubility.The initial concentration of the stock solutions was 2 mg/ml for all samples.All compounds were stored at −20˚C until testing.Ementine was used as the positive control in all experiments.The initial concentration of all samples were 100 mg/ml, which was serially diluted in complete medium with 10-fold dilutions to give 6 concentrations, the lowest being, 0.001 μg/ml.
The 50% inhibitory concentration (IC 50 ) values for these samples were obtained from dose response curves, using a non-linear dose response curve fitting analyses via GraphPad Prism v, 4.0 software.

Results and Discussion
Column chromatography of the acetone leaf extract using dichlomethanehexane solvent system mixtures yielded known methoxylated flavonoids 1 and 3 (Figure 1). 1 H NMR and 13 C NMR spectra are shown on Figures 2-5.

Cytotoxicity Screening Tests
Compounds 1 and 3 had no significant cytotoxic effect against the CHO cell-line as observed in Table 1 and Figure 12 on dose-response curves.
Figure 12.Dose-response curves of fractions using the CHO cell-line.
Table 1.IC 50 -values of fractions using the CHO cell-line.The propensity of a flavonoid to inhibit free-radical mediated events is governed by its chemical structure.The number of substituent groups and their positions on the flavonoid structure can influence the radical-scavenging activity [27].

Conclusion
The present study indicate that 5-hydroxy-7-methoxyflavanone (pols 1) and 5-hydroxy-6,7-dimethoxyflavanone (pols 3) are effective anti-oxidants in retarding the formation of primary and secondary oxidation products during deep frying using vegetable oils.The two flavanones had no significant cytotoxic effect and are therefore potential food additives intended to serve as antioxidant principles in oil deep frying processes and in the formulation of animal feeds.The

Figure 6 .
Figure 6.Changes in p-AV for plain oils heated to 180˚C -200˚C.

Figure 7 .
Figure 7. Changes in peroxide values for plain oils heated to 180˚C -200˚C.

Figure 10 .
Figure 10.Changes in p-AV values for Sunflower oil heated with 100 ppm of compounds 1 and 3.

Figure 11 .
Figure 11.Changes in peroxide values for Sunflower oil heated with 100 ppm of compounds 1 and 3.

Chromatographic Methods 20
g of the extract was introduced into a glass column (4 cm diameter), packed with 129 g of 230 -400 mesh silica gel.The column was sequentially eluted with 500 ml portions of hexane, 40% CH 2 Cl 2 in hexane and finally with 60% CH 2 Cl 2 in hexane, effectively eluting four coloured bands ranging from yellow to red.
[20].Wanjau et al.DOI: 10.4236/fns.2018.9110911275FoodandNutritionSciences clear of the orange exudate.The acetone extract was decanted and filtered through a filter paper into a 2.5 L brown glass bottle.The acetone was recovered using a rotary evaporator at a rotate speed of 100, heated at a water bath regulated at 60˚C.The concentrate was preserved in an open brown glass bottle (100 ml) and stored in a vacuum desiccator until it gave a constant mass of 47.5 g (i.e.20.2% of dry leaves) on weighing.2.3.Identification of the compounds was achieved through correlation of spectral data, melting point values and comparison with literature data[20].