Because of their high polyphenol content, fruit and vegetable by-products have lately been proposed as natural food additives. Total phenolic content was calculated using the Folin-Ciocalteu method, and antioxidant capacity was determined using DPPH Radical-Scavenging Assay (RSA).
Pomegranate peels showed the highest values of total phenolic content 212.3 mg GAE/g do and antioxidant capacity of 95.7 of inhibition percentage for the DPPH assay. Staphylococcus aureus and Pseudomonas fluorescens were the bacteria that were most inhibited by it.
They are of research interest because of their perceived health benefits. They exhibit many effects such as anti-carcinogenic, anti-atherogenic, anti-ulcer, anti-thrombotic, anti-inflammatory, analgesic effects, etc. They are also used as antioxidants in the food industry. They provide beneficial effects because of free radical scavenger properties.
The basic principle of the FC method is oxidation/reduction reaction. The FC reagent contains phosphomolybdic/phosphotungstic acid complexes. This method relies on the transfer of electrons in an alkaline medium from phenolic compounds, to form a blue chromophore. The Blue chromophore is constituted by phosphomolybdenum /phosphotungstic acid complexes. The reduced Fc reagent is detectable in the 690-710 nm range. The maximum absorption depends on the concentration of phenolic compounds present in a sample.
1. Gallic acid
2. Stock concentrated : 1mg/ml
We can make it 10 or 100 ml according to the requirement of the experiment by dissolving the gallic acid in the distilled water
20% Sodium Carbonate
Because this rxn is carried out in basic medium
Dissolve Sodium Carbonate by shaking the container
FC Reagent: It is always stored in dark.
Any solvent can be used for plant extraction.
Ex Methanolic, Ethanolic, acetonic plant extracts.
Add 100 microL of plant extract into an Eppendorf tube.
Add 400 microL of distilled water to dilute the sample.
Add 150 microL of FC reagent (diluted 1:1)
Leave it for 5-10 min at room temp.
A bluish color compound is formed.
Add 500 microL of 20% sodium carbonate
Hold it for 60 min in dark.
Then take the absorbance
Take different concentrations Of standard gallic acid i.e. 50, 100, 150, 200, 250, up to 500 microL.
The stock concentration used is 1mg/ml.
Now make up all test tubes up to 500 microL with distilled water.
Add 150 microL of FC reagent to all the test tubes.
Test tubes will have a gradual increase in greenish color intensity in test tubes due to an increase in phenolic content of the standard.
Add 500 microL of Sodium Carbonate to all tubes.
Hold them for 60 min in dark.
Then take the absorbance.
All the above chemicals are added in the same concentration and volume except sample or standard.
DPPH stands for 2,2-diphenyl-1-picrylhydrazyl. DPPH is a dark-colored crystalline powder that is composed of stable free-radical molecules. But most importantly it’s used to monitor chemical reactions that involve radicals Like in the case of antioxidant assay. DPPH is a popular radical and a very efficient scavenger for other radicals.
DPPH assay is used to measure the antioxidant activity of molecules that have the ability to transfer electrons. The compound is actually a chromophore which is a stable radical cation possessing purple color. The radical cation shows maximum absorbance at 517 nm.
So Antioxidant compounds, having the capacity to donate an electron to DPPH radical cation, cause a discoloration of the solution when added to the solution having DPPHradical in it. The disappearance of color or simply change in color is estimated spectrophotometrically
Preparation of DPPH solution
7.89 of DPPH is weighed on a chemical balance.
Dissolve this DPPH in 99.5% ethanol to get a constant volume by filling 100 mL of a measuring flask.
The absorbance of the DPPH solution decreases with time until around 1hr after preparation.
The solution is kept in dark for 120 min to stabilize the absorbance.
Add 200 microL of analytical sample and also 800 microL of 0.1 M Tris-HCl buffer having pH set at 7.4. After 120 min period is over.
Add 1 mL of DPPH solution to this tube. Then absorbance is taken at 517 nm.
Similarly, One more test tube with 1 mL DPPH solution and 800 microL of 0.1 M Tris-HCl, but instead of adding an analytical sample, we add 200 microL of ethanol into this tube.
A blank tube is prepared by mixing 1.2 mL of ethanol and 800 microL of 0.1 M Tris-HCl buffer
Sample collection and preparation of pomegranate peels extract
The peels of pomegranate were cleaned and cut into small pieces, and then oven dried at 50oC for 48 h. The dried sample was then pulverized using a mechanical grinder and passed through a 250 μm mesh and then stored at 4oC until use. The different types of solvents used were absolute methanol, ethanol, acetone, water, and their aqueous solutions at 50% and 80% concentrations.
Total phenolic content (TPC)
The amount of total phenolic content (TPC) in pomegranate was determined with the Folin-Ciocalteu reagent base.
About 0.5 mL of Folin-Ciocalteu reagent (10%, v/v) was added to 0.1 mL of pomegranate extract sample.
The mixture was swirled and allowed to stand for 6 min followed by the addition of 1 mL 7.5% (w/v) of sodium carbonate (Na2CO3) and samples were mixed.
Solutions were allowed to stand for 2 h at room temperature in dark and the absorbance was read at 765 nm wavelength using a spectrophotometer.
Gallic acid standard solutions were prepared by dissolving gallic acid in water at concentrations ranging from 0 to 250 mg/L.
The results were expressed as milligrams of gallic acid equivalents per 100 g of sample (mg GAE/100 g of DW).
All the measurements were taken in triplicates and mean values were calculated.
RESULT AND DISCUSSION
The TPC of Pomegranate Peel was found to be 212.3 mg of GAE/g dw.
Pomegranate (peel) 197.1±1.76g,B 173.2±3.54f,A 212.3±3.31g,C
The TPC of pomegranate peel aqueous acetone extracts (212.3 mg GAE/g dw) was
nearly 16-fold higher than that of aril pomace extracts (13.2 mg GAE/g dw)
Larger amounts of phenols were found in pomegranate
peel with respect to arils (249.4 mg GAE/g and 24.4 mg GAE/g dry extract, respectively).
Pomegranate Peel could be considered a polyphenol-rich product.
DPPH Radical-Scavenging Assay (RSA)
The 2,2-diphenyl-1-picrylhydrazyl was dissolved in methanol to prepare the DPPH solution.
The DPPH solution was diluted several 42 times with methanol to obtain 0.9 absorbances at 516 nm, using a spectrophotometer.
1 ml of DPPH solution was added to 100 μl of pomegranate extract solution.
The mixture was shaken in a vortex and kept for 2 h in a dark place.
After 2 h, the mixture was transferred to microplate plastic and absorption of DPPH solution after the addition of the sample was measured at 516 nm using the spectrophotometer.
The change in absorption of each sample is computed as the difference between the blank and sample readings.
The percentage of DPPH scavenging activity was calculated using the following equation:
Radical scavenging (%) = [(A0 – A1 / A0) × 100]
Where A0 is the absorbance of the control and A1 is the absorbance of the sample extracts.
What if Folin–Ciocâlteu reagent?
The Folin–Ciocâlteu reagent (FCR) or Folin’s phenol reagent, also called the gallic acid equivalence method (GAE), is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric in-vitro assay of phenolic and polyphenolic antioxidants.
How does DPPH radical scavenging activity work?
DPPH free radical scavenging is an accepted mechanism for screening the antioxidant activity of plant extracts. In the DPPH assay, violet color DPPH solution is reduced to a yellow-colored product, diphenylpicryl hydrazine, by the addition of the extract in a concentration-dependent manner.
RESULT AND DISCUSSION
Primary and secondary antioxidants are the two types of antioxidants. As these antioxidants react to scavenge free radicals from the DPPH solution, they suppress the formation of the free radical initiation chain and disrupt the propagation chain by donating hydrogen atoms or electrons, DPPH assays are frequently used to measure the capability of primary antioxidants in plants.
Free radicals are converted into a more stable product in this process. The DPPH solution turns from purple to yellow as a result of this circumstance. Depending on the type of solvent utilized, the DPPH scavenging percentages for pomegranate peel ranged from 45 to 88.46 percent.
The results of this study showed that the type of solvent used had a significant effect (P<0.05) on the extraction of antioxidant compounds from pomegranate
TPC and DPPH values of pomegranate peel extracts decreased with an increase in the organic solvent concentration.
In fact, it can be concluded that the extracts obtained using higher polar solvents were more effective than fewer ones.
The addition of 50% water to methanol, acetone, or ethanol can enhance the extracting power and antioxidant activity estimation, especially methanol and acetone.
The phenolic compounds TPC assay showed a good correlation with antioxidant activity DPPH of pomegranate peel.