1/3 15.3b Natural Product Screening- Anti-oxidant screen of extract Crude Extract

Protocol No. & Title: 15.3b Natural Product Screening: Anti-oxidant Screen for Extracts

Version Date: 5November 2012: Version 2

Author: Dr. Marsha J. Lewis

Purpose: This is known as a standard 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay. The DPPH assay is popular in natural product antioxidant studies. One of the reasons is that this method is simple and sensitive. This assay is based on the theory that a hydrogen donor is an antioxidant. It measures compounds that are radical scavengers. Figure 1, below, shows the mechanism by which DPPH accepts hydrogen from an antioxidant. DPPH is one of the few stable and commercially available organic nitrogen radicals (1). The antioxidant effect is proportional to the disappearance of DPPH in test samples. Monitoring DPPH with a UV spectrometer has become the most commonly used method because of its simplicity and accuracy. DPPH shows a strong absorption maximum at 517 nm (purple). The color turns from purple to yellow followed by the formation of DPPH upon absorption of hydrogen from an antioxidant. This reaction is stoichiometric with respect to the number of hydrogen atoms absorbed. Therefore, the antioxidant effect can be easily evaluated by following the decrease of UV absorption at 517 nm.

Figure 1. DPPH free radical conversion to DPPH by anti-oxidant compound.

References: 1. MacDonald-Wicks, L. K.; Wood, L. G.; Garg, M. L. Methodology for the

determination of biological antioxidant capacity in vitro: a review. J. Sci. Food Agric. 2006, 86, 20462056.

2. Moon, J. K.; Shibamoto, T., Antioxidant assays for plant and food

components. Journal of agricultural and Food Chemistry 2009, 57, (5), 1655-1666.

Materials: Following solutions should be in lab stock: 100mM Tris-HCL pH 7.4 and 10 mg/ml -tocopherol (positive control, Vitamin E)

Read through protocol to prepare other materials

Safety Notes: 1. Wear gloves, lab coat, and goggles as needed. Work in fume hood as required.

2/3 15.3b Natural Product Screening- Anti-oxidant screen of extract Crude Extract

Prepare extract or purified fraction for testing

You need at least 1 mg (that is milligrams---not grams!) of product for testing.

How do measure 1 mg of extract? You should have a known amount of extract that you weighed after isolation (X mgs) and you should have added a known amount of solvent for temporary storage (e.g. 0.5 ml (a.k.a. 500 l) methylene chloride). So, you have a solution that you know is X mgs of extract per ml (in glass vialhopefully, you did NOT use plastic or the plastic could degrade in your solvent). One word of warning: if you used a very volatile solvent for dissolution of your isolated product, it will evaporate, even in cold storage. To ensure you know the correct concentration of isolated product, make sure you have the volume that you think you have by measuring the volume with a pipettor or mark the bottle when you first dissolve your product so you know the initial volume. As the solvent evaporates, your isolated product may also begin to come out of solution. Add back solvent to completely solubilize or to bring to the concentration you desire.

Then, measure 1.0 mgs for this assay preparation:

Use the rotary evaporator to remove the solvent from your 1.0 mgs of extract, unless your product

is dissolved in methanol. After the solvent is removed, add 500 l of methanol. Your extract should

be soluble in 100% methanol. If it is not, let me know. Your concentration is 2 mg of extract

product/ml of methanol. You can prepare this a day or two ahead of time and store in cold storage.

1. Prepare 12 mls of 0.1 mM DPPH solution with methanol. DPPH molecular weight is 394.32 g/mol.

You should calculate 0.5 mg of DPPH is required to make 5 mls of 0.1 mM DPPH solution. DPPH is

stored in the freezer. It should be protected from light and the time out of the freezer should be

minimized. DPPH cost is approximately $100/gram. (Be aware you only need 0.5 mg or 0.005

grams! Hint: To minimize DPPH losses, measure DPPH directly to the container you will prepare the

solution so you do not need to transfer 0.005 g from weighing paper to container. )

2. Add 0.005 g of DPPH to 12 mls of methanol measured with a graduated cylinder into a small flask

wrapped in foil to protect the solution from light.

3. Assemble eleven (11) two ml microcentrifuge tubes and label as follows:

a. Tubes 1a-c through 3a-c: Product Extract Dilution 1 through 3 (repeat three times for 9

tubes)

b. Tube 4: Positive control, -tocopherol

c. Tube 5: Negative control, solvent only

4. Prepare your positive control, -tocopherol (chemical structure below in Figure 2). The stock solution

is at 10 mg/ml (in ethanol, stored in the refrigerator, and protected from light). Remove 10 l from

the -tocopherol stock and place in a labeled eppendorf, on ice and shielded from light. 10 l of

10mg/ml stock is 100g of -tocopherol. Add 40 l of 100% ethanol to the positive control tube.

3/3 15.3b Natural Product Screening- Anti-oxidant screen of extract Crude Extract

Figure 2. The chemical structure of -tocopherol (fat soluble Vitamin E). -tocopherol is insoluble

in water.

5. Add 50 l of prepared extract at 2 mg/ml to Tube 1 a-c (total of 100 g of extract)

6. Add 38 l of prepared extract to Tube 2 a-c (total of 75 g of extract)

7. Add 25 l of prepared extract to Tube 3 a-c (total of 25 g of extract)

8. Add 50 l of methanol only to Tube 5, your negative control.

9. Add 450 l of Tris-HCl buffer (pH 7.4) to Tube 1 (a-c) and your positive (tube 4) and negative (tube 5)

controls.

10. Add 462 l of Tris-HCl buffer (pH 7.4) to Tube 2 (a-c).

11. Add 475 l of Tris-HCl buffer (pH 7.4) to Tube 3 (a-c).

12. Add 1 ml of the prepared 0.1 mM DPPH solution to all the tubes.

13. Incubate all the tubes in the dark for 30 minutes at room temperature.

14. After 30 minutes, read the absorbance at 517 nm for each sample on the NanoPhotometer.

a. Power on.

b. Select 3) Functions

c. Select 1) Single Wavelength

d. Enter 517 nm for the wavelength

e. The mode should be Absorbance

f. The pathlength should be 10mm

g. Use the 1 ml disposable cuvettes. Blank with 1 ml of water.

h. Analyze product samples (nine) and positive and negative controls.

15. Record data. The negative control should equal the blank. All data is converted into percentage of

scavenging radical.

The triplicate data should be averaged and standard deviation determined.