AAL’s “Natural Products Biochemistry Department” has the capability for testing antioxidant, free radical scavenging activity on natural products such as fruit, berries, vegetable, and herbal products.
List of AAL Antioxidant Activity Services:
- 1. Total Anthocyanin as Cyanidin
- 2. Total Polyphenols as Gallic Acid Equivalent (GAE)
- 3. Total Flavonoid as Catechin Equivalent
- 4. Total Proanthocyanidin
- 5. FRAP Ferric Reducing Antioxidant Activity
- 6. DPPH
- 7. TEAC
- 8. ORAC
8a. Lipophilic (Oil Soluble)
8b. Hydrophilic (Water Soluble)
Anthocyanins are natural colorant flavonoids of fruits and vegetables that have deep colors. Examples of anthocyanins are the acai berry, grapes skin, grapeseed, bilberry, maqui berry, blueberry, cranberry, or pomagranate juice extract. Anthocyanin are very powerful antioxidants that benefit the entire body by protecting the cells from free radicals. The most common anthocyanin groups are aglycone of cyanidin, delphinidin, malvidin, petunidin, and pelargonidin.
Determination of anthocyanidins is performed by alcohol extraction of leaves, petals, seeds, berries, or herbs. A measurement of absorbance is taken from two difference pH solutions and measured at two different wavelengths; this is known as the pH differential method. The total anthocyanins are reported as cyn 3-glu per 100 grams.
Only the natural color anthocyanins from fruit or vegetable sources are pH sensitive in acidic conditions. An increase in spectrophotometry absorbance is observed when there is a change in acidity in the fruit or vegetable during the anthocyanin test. On the other hand, any artificial red or purple color may be discolored when extracted with alcohol in acidic conditions, and may show no change in color or no response to the anthocyanin test.
Phenolic compounds in natural products are one or multiple hexagonal rings with three double bonds inside the ring, and more than one hydroxyl or OH group attached to it. Examples include chlorogenic acid, quercetin, gallic, flavonoid, etc.
The TP method uses gallic acid as phenolic standard, gallic acid ug/mL at different concentrations ug/mL, and for linearity curve and extracted samples, are determined simultaneously by colorimetry with the Folin–Ciocalteu method. Results are expressed as gallic equivalent per gram sample.
The flavonoids from fruit and vegetable products are extracted, and using catechin as a standard, the total flavonoids in food products are measured by aluminum chloride reduction reaction. The absorbance is measured against a reagent blank at visible spectrometry. Total flavonoid content is expressed as a percentage of catechin equivalent per 100 gram sample mass.
Proanthocyanidins or oligomeric proanthocyanidins (OPCs) are phenolic organic clusters of dimers (2), trimers (3), tetramers (4) and pentamer natural compounds in fruits and vegetables. OPCs are determined by AOAC–USDA DMAC method. Using procyanidin B2 as a standard, reported result as mg of proanthocyanidin per gram sample.
Sample and sample blank are treated with Fe+3, TPTZ, presence of antioxidants show a reduction of Fe+3 to Fe+2 show increase of blue ferrous form. Change in absorbance is proportional to reducing/ antioxidant power(FRAP), and expressed as micromole trolox equivalent per gram sample mass (umol TE/g).
DPPH measures the ability of bioactive to act as free radical scavengers, or hydrogen donors, using trolox as reference standard. The odd electron in the DPPH free radical gives a strong absorption purple color. Upon reacting with the bioactive chemical, the odd electron in the DPPH radical becomes paired with the hydrogen from the free radical scavenger antioxidant, and the color turns from a purple to yellow. The more discolorized of DPPH solution after reaction, the higher the antioxidant scavenger or umole-trolox unit per 100 grams.
TEAC method is based on the suppression of the absorbance of radical probe (ABTS 2,2′-azinobis (3-ethylbenzothiazoline 6-sulfonate)) by antioxidant sample, when ABTS is incubated with peroxidase and H2O2.
Hydrophilic ORAC measures watersoluble antioxidant activity in the natural product sample such a fruitor vegetable. Using AAPH as peroxy radicals generator and fluoresceinas reaction proble observe the decay of the fluorescence emissioncurve, using Trolox as standard.
Lipophilic ORAC measures oilsoluble antioxidant activity. Samples are extracted with buffer, andhexane. The hexane layer is used for L-ORAC. Using pAAPH as peroxyradical generator, fluorescein as as reaction probe, observed the oilsoluble decay under fluorescen emission curve, using Trolox asstandard.