Note: This project uses materials and equipment found in a biochemistry laboratory. The student will need to work with a mentor who is a biochemistry research scientist or graduate student who can assist with performing certain procedures and operating various types of equipment.
To determine the amount of antioxidants present in various teas and compare the concentration of antioxidants of the first flush of tea to two subsequent flushes with the same tea leaves or tea bag.
Free radicals have been known to cause a multitude of health problems and diseases through cell damage and the disturbance of DNA. Free radicals are atoms or groups of atoms with one or more (unpaired) electrons. Antioxidants, conversely, are molecules that can safely interact with free radicals and end their cumulative destruction before vital molecules are damaged—by donating electrons to the unpaired electrons found in the free radicals. Tea is believed to contain a large amount of antioxidants, and people in some cultures believe that subsequent flushes with the same tea leaves or tea bag are more beneficial to one's health. This project will test the validity of these beliefs.
- green tea, oolong, and black tea bags/leaves
- distilled water
- hot plate
- FisherTM p8 filter paper
- F-C reagent
- 2% NaCo3 solution
- warm water bath
- gallic aci
- UV/Visible spectrophotometer
Tea samples will be prepared and tested for antioxidant presence. Initially, the total amount of polyphenols present in each tea sample will be determined. (Polyphenols are naturally occurring chemical compounds that are powerful antioxidants. In tea, the primary polyphenols are called catechins. Other polyphenols found in tea include flavanols, flavanol glycosides, flavandiols, phenolic acids, and depsides.) This will be done using the Folin-Ciocalten assay test. A UV/visible spectrometer will be used for absorbance measurements to determine the amount of antioxidant in each sample.
The Folin-Ciocalten (F-C) assay test for determining total amount of polyphenols in each tea sample.
- Choose one of the teas to be studied. Pour a measured amount of loose tea leaves or place a tea bag (of this particular type of tea) into a beaker and then pour in 100mL of boiling distilled water. Cover, and after 5 minutes separate the tea from the tea bag or from the tea leaves by filtering it through p8 filter paper. Repeat this procedure two more times with the same tea leaves or tea bag to obtain a total of three flush samples.
- In a clean beaker, take the pipettes and dilute 0.27ml tea extract solution from step 1 in 15.63 ml water, 0.1 ml ethanol, 1.00 ml F-C reagent, and 3.00 ml 2% NaCO3 to make a total of 20.00 ml. Heat at 50°C in a water bath for 5 minutes. Determine the absorbance on a UV/visible spectrophotometer at 765 nm. Repeat steps 1–2 two more times using the other flush samples from the same tea leaves or tea bag.
- Prepare a calibration curve using gallic acid as the standard of phenol at concentrations of 1, 2, 3, 4, 5, × 105M. Compare the absorbance of the sample with the calibration curve to obtain the total polyphenol content of each tea sample from the same tea leaves or tea bag.
- Repeat steps 1–3 with the remaining tea samples.
- What were the total amounts of polyphenols present in each tea sample after the first flush? What were the total amounts of polyphenols present in each tea sample after the second and third flushes?
- Which tea type contained the greatest amount of polyphenols after the first flush? After the second flush? After the third flush?
- Which tea had the greatest concentration of polyphenols overall?
- Assuming that an increased presence of polyphenols found in tea is beneficial to one's health, which tea and which flush of this tea proved to have the highest concentration of polyphenols?