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A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes
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Summary June 10th, 2022
Phenolic acids are important phytochemicals that are present in whole grains. They possess bioactive properties such as antioxidant protective functions. This work aimed at reporting on a generalized method for the HPLC identification, total phenolic content estimation, and determination of the antioxidant capacity of phenolic acids in cereals and legumes.
Transcript
A generalized method for determining free soluble phenolic acid composition and antioxidant capacity of cereals and legumes. Whole grains, including cereals and legumes, form a substantial part of human diets. Their nutritional relevance to humans has long been recognized.
However, more recently, their antioxidant protective health benefits have been reported. Phenolic acids found in the outer grain layers of cereals and the seed coat of legumes contribute to the antioxidant properties of whole grains. They scavenge free radicals that cause oxidative damage to biomolecules.
The two classes of phenolic acids found in whole grains are hydroxybenzoic acids and hydroxycinnamic acids. This study provides a simple method for extracting whole grain phenolic acids and determining their in vitro antioxidant capacity. Use five whole grain samples for this study, durum wheat, yellow corn, black-eye cowpea bean, soybean, and red kidney bean.
Accurately weigh 100 milligrams of the whole grain flour sample directly into an amber-colored, two milliliter capacity micro centrifuge tube. The dark color of the tube helps to prevent exposure of the mixture to light. Add one milliliter of 80%aqueous methanol to each of the tubes containing examples.
Vortex briefly to mix the methanol solution and sample. Sonicate the samples for 60 minutes to extract the free soluble phenolic compounds. Put a cover over the samples for the duration of sonication for added protection from light.
After sonication, centrifuge the mixtures at 20, 000 times G for five minutes to sediment the solid residues, leaving the supernatant on top. Free phenolic compounds will be present in the supernatant after centrifugation. The supernatant needs to be filtered prior to injecting into the HPLC instrument.
To filter the supernatant, remove the plunger of a 3 mL syringe and attach a syringe filter. The filter should have a pore size no larger than 0.22 micrometers. Pipette approximately 0.4 milliliters of the supernatant into the top of the syringe.
Reinsert the plunger and push the liquid through the filter into an HPLC vial containing a vial insert. Once the instrument has been set up to run the method outlined in the manuscript for HPLC analysis, load the vials into the carousel to correspond with the sample list. Obtain HPLC chromatograms at 320 nanometers and 280 nanometers, showing distinct peaks representing different phenolic compounds.
Using appropriate standard curves, quantify hydroxycinnamic acids at 320 nanometers since they have a maximum absorbance at this wavelength. By the same principle, quantify hydroxybenzoic acids at 280 nanometers. Use Trolox, a water soluble analog of vitamin E, as a standard to estimate the in vitro antioxidant capacity of the whole grain extracts.
Accurately weigh one milligram of Trolox into a Falcon tube. Dissolve with four milliliters of 50%aqueous methanol to prepare a stock solution of one millimole per liter, which is the same as 1000 micromole per liter. Prepare six concentrations of Trolox that is 50, 100, 200, 400, 600, and 800 micromole per liter to plot standard curves for the estimation of DPPH radical scavenging capacity and Trolox'equivalent antioxidant capacity, TEAC.
Similarly, prepare 6.25, 12.5, 25, and 50 micromole per liter Trolox concentrations for estimating oxygen radical absorbance capacity, ORAC. Make up the total volume of each concentration to 500 microliters, as shown in table one. Dilute the sample extracts with methanol prior to analysis.
Here, yellow corn and cowpea extracts were diluted two times. The wheat and kidney bean extracts were diluted five times, while the soybean extract was diluted 10 times with methanol. Weigh 8.23 milligrams of ABTS into a clean, two milliliter capacity, amber micro centrifuge tube.
Next, weigh 1.62 milligrams of potassium persulfate into a clean, two milliliter capacity, amber micro centrifuge tube. Dissolve each of the weighed chemicals in one milliliter distilled water by vortexing. This results in a 16 millimolar ABTS solution and a six millimolar potassium persulfate solution.
Prepare the ABTS stock solution by mixing the ABTS and potassium persulfate solutions in equal volumes. The solution will immediately change to a dark color. Allow this mixture to incubate in darkness for 12 to 16 hours.
Dilute the ABTS stock solution 30 times with 200 millimolar phosphate-buffered saline to form the ABTS working solution. To do this, add 58 milliliters of 200 millimolar PBS to two milliliters of the ABTS working solution. The working solution will contain 0.27 millimolar ABTS and 0.1 millimolar potassium persulfate.
For the analysis, place 10 microliters of each diluted extract in a 96 well microplate. Add 190 microliters of ABTS working solution to each well and incubate for 60 minutes. Measure the absorbance of the reaction mixtures at 750 nanometers in a microplate reader.
Use the Trolox standards in concentrations ranging from 100 to 800 micromole per liter to plot a calibration curve. The DPPH antioxidant assay requires a radical-generating compound, DPPH. Weigh out 1.2 milligrams of DPPH into an empty 15 milliliter capacity centrifuge tube.
Dissolve the DPPH in methanol to prepare a 60 micromolar solution. The DPPH assay tests the ability of the sample extracts to scavenge free radicals produced by DPPH. Add five microliters of sample extract into the microplate wells.
Next, add 195 microliters of 60 micromolar DPPH methanolic solution and incubate for 60 minutes. Measure the absorbance at 515 nanometers. Use the Trolox standards to plot a calibration curve.
Figure one shows the structure of hydroxybenzoic acids found in the whole grains. In this current study, vanillic acid was the only hydroxybenzoic acid identified. Figure two shows the structure of hydroxycinnamic acids found in whole grains.
In the current study, p-coumaric, caffeic, and ferulic acids were identified in the samples. Table two shows the phenolic acids identified in the samples. As mentioned earlier, vanillic acid was the only hydroxybenzoic acid identified in the samples.
It was identified in the cowpea extract only. The hydroxycinnamic acid caffeic acid was identified only in kidney bean, while p-coumeric acid was identified in yellow corn, cowpea, and soybean. Ferulic acid was identified in all of the samples and was the predominant phenolic acid in the samples.
The total concentration of the phenolic acids in order from greatest to least was in the order soybean, cowpea, yellow corn, and kidney bean or equivalent, followed by wheat. Table three showed the total phenolic content and antioxidant capacity of the samples. The antioxidant capacity comprised DPPH radical scavenging capacity, TEAC, ORAC, and total antioxidant capacity of the samples.
The total antioxidant capacity is the sum of the DPPH, TEAC, and ORAC values. Similar to table two, soybean had the highest total antioxidant capacity. However, instead of cowpea, it was rather kidney bean that had the second highest total antioxidant capacity, although cowpea had the second highest total phenolic acid content.
This anomaly is related to the structures of the individual phenolic acids and the possible antagonistic effect of the hydroxybenzoic acid vanillic acid on the antioxidant effects of the hydroxycinnamic acids in cowpea. This study concluded that whole grains differ in their phenolic acid compositions. Among the whole grains studied, soybean has the highest total amount of phenolic acids and, correspondingly, the highest antioxidant capacity.
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