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1/3 15.3b Natural Product Screening- Anti-oxidant screen of extract Crude Extract
Prepare extract or purified fraction 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.
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.
2/3 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.
3/3 15.3b Natural Product Screening- Anti-oxidant screen of extract Crude Extract