We analyzed OXYFOX^tm for antioxidant activity using a Metrohm 679 Rancimat 1. The major phenolic compounds acting as antioxidants were identified by LC-MS. We also analyzed the sample for levels of the phytoestrogens genistin and genistein by HPLC.

This study showed that OXYFOX^tm was found to contain 0.4% phenolic compounds. The study also showed that 0.02% of the acidified ethyl acetate extract of this sample was a more effective antioxidant than 0.02% butylated hydroxytoluene (BHT). The primary phenolic compound imparting antioxidant potential in this sample was identified by LC-MS as kaempferol. The concentrations of genistin and genistein were 10 ppm and 2 ppm respectively.

Dr. Richard Hiserodt, Ph.D. at Rutgers University, Center for Advanced Food Technology commented about this study on OXYFOX^tm that, ". . . the antioxidant levels are high, showing potential nutraceutical value."

ANALYTICAL METHOD

1. The sample was extracted with methanol.
2. The residue from the methanol extract was concentrated almost to dryness using a rotary evaporator at 40o C.
3. It was then concentrated to dryness using a stream of nitrogen at room temperature.
4. The residue from the methanol extract was extracted with acidified ethyl acetate and the extract concentrated to dryness using a rotary evaporator at 40o C.
The sample weight of OXYFOS^{tm was 26.5 grams. This yielded 17% methanol extract and finally 0.4% acidified ethyl acetate extract (phenolic compounds).
1 Manufactured by Metrohm Ltd., Herisau - Switzerland}

The acidified ethyl acetate extract of OXYFOS^tm was added to lard and analyzed for antioxidant activity using the Metrohm 679 Rancimat. This instrument measures the rate of oxidation of fats by bubbling dry air (20 L/min.) through the heated sample (110o C.) and measuring the liberated volatile oxidation products using a conductivity cell. The time to complete oxidation of the sample is the induction time. The acidified ethyl acetate extract of OXYFOS^tm was added to lard at the 0.02% level and compared to lard with 0.02% BHT and to lard without BHT. Typical induction times for lard with 0.02% BHT and lard without BHT are, 6.8 hrs. and 2.3 hrs. respectively.

The acidified ethyl acetate extract of OXYFOS^tm was analyzed by LC-MS to identify the compound or compounds responsible for its good antioxidant activity. The components in this extract were separated using a TosoHaas ODS-8OTm, 5 m, 250 mm x 4.6 mm (i.d.) analytical column with gradient elution. The mobile phase consisted of water with acetonitrile as the organic modifier. A Varian 9012 HPLC Solvent Delivery System was interfaced with VG Platform II Mass Spectrometer. Mass spectral data was acquired by atmospheric pressure chemical ionization (APcI) in the positive and negative ion mode.

The LC-MS data showed the major phenolic compound in the acidified ethyl acetate extract of OXYFOS^tm to be kaempferol. HPLC analysis with UV-detection at 254 nm showed kaempferol to be the major phenolic compound in OXYFOS^tm.

Semi-quantitative levels of genistein and genistin were determined by HPLC using the smne instrument conditions outlined above but with UV-detection at 254 nm. The concentrations of genistin and genistein were 10 ppm and 2 ppm respectively.