Pharmaceutical Chemistry 127.

1
Section OS8

Experiment 6
IN VITRO ANTIOXIDANT ACTIVITIES OF VITAMIN C AND VITAMIN E

I. Introduction

Free radicals, molecules with unpaired electrons, are produced when there is an
oxidation of certain molecules. Most free radicals in the biological system are derivatives of
oxygen (Reactive Oxygen Species or ROS) and nitrogen (Reactive Nitrogen Species or RNS).
They are highly reactive and causes chain reactions that may cause damage or death of cells.
Some of the cellular macromolecules that are susceptible to free radical damage include lipids,
proteins and nucleic acids.

Antioxidants help in neutralizing free radicals by donating or accepting an electron to

eliminate the unpaired condition, consequently inhibiting oxidative chain reactions. They act by
scavenging reactive oxygen species, by reduction, by inhibiting free radical formation, by
binding transition metal ions and preventing formation of hydroxyl radicals and/or decomposition
of lipid peroxidases, by repairing damage or any combination of the above.

Sources of antioxidants may be endogenous and exogenous. Endogenous antioxidants

include gluthatione, aplha-lipoic acid, coenzyme Q, ferritin, uric acid, bilirubin, metallothioneine,
L-carnitine and melatonin. Exogenous antioxidants, on the other hand, are those found from the
diet, including fruits and vegetables or from dietary supplements.

There are several methods in determining the antioxidant properties of substances. In

this experiment, the DPPH assay was used to measure the free-radical scavenging activities of
Vitamins C and E. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable, free radical that contains an
odd electron and produces a violet solution in ethanol. In the presence of an antioxidant
molecule, the DPPH radical is reduced and decolorized. The DPPH assay provides an easy and
rapid way to quantitatively determine the changes in absorbance among the different
concentrations of the antioxidant used through spectrophotometry.

II. Objectives
- To explain the different possible mechanisms of antioxidants and their roles as defense
mechanisms in the body
- To enumerate different methods of determining in vitro and in vivo antioxidant activities
- To determine the in vitro antioxidant activities of vitamin C and vitamin E
III. Procedure
A. DPPH assay
To make different concentrations of ascorbic acid, 10.0 ml, 5.0 ml, 2.0 ml and 1.0 ml of
each of the stock solutiondiluted to 50 ml. To 2.0 ml of each concentration, 2.0 ml of 0.30 mM
DPPH was added and allowed to react in the dark for 20 minutes. The absorbance of the
different concentrations and the negative control (2.0 ml of 0.30 mM DPPH with 2.0 ml ethanol)
were measured at 517 nm. The percent inhibition (%Inh) for each concentration was calculated
using the formula:
%Inh= (Ao-Ac)/Ao

B. Thiobarbituric Acid – Reactive Substances (TBARS) Assay

(INSERT PROCEDURE)

IV. Results and Discussion

Diphenylpicrylhydrazyl is characterized as a stable free radical by virtue of the

delocalization of the spare electron over the molecule as a whole, so that the molecules do not
dimerise, as would be the case with most other free radicals. The delocalization also gives rise
to the deep violet colour, characterised by an absorption band in ethanol solution centred at
about 520 nm.

When a solution of DPPH is mixed with that of a substance that can donate a hydrogen
atom (antioxidant), then this gives rise to the reduced form (2) with the loss of this violet colour
(although there would be expected to be a residual pale yellow colour from the picryl group still
present). Representing the DPPH radical by Zo and the donor molecule by AH, the primary
reaction is:
Zo + AH = ZH + Ao [1]

where ZH is the reduced form and Ao is free radical produced in this first step. This latter radical
will then undergo further reactions which control the overall stoichiometry, that is, the number of
molecules of DPPH reduced (decolorized) by one molecule of the reductant. The reaction [1] is
therefore intended to provide the link with the reactions taking place in an oxidizing system,
such as the auto-oxidation of a lipid or other unsaturated substance; the DPPH molecule Zo is
thus intended to represent the free radicals formed in the system whose activity is to be
suppressed by AH which is the antioxidant.

In this experiment, DPPH assay was used to test for the determination of the properties
like the percent inhibition of the antioxidants specifically ascorbic acid and alpha-tocopherol.
Increasing the amount of antioxidant would therefore decrease the absorbance values. For
each antioxidant, the concentration (x-axis) was plotted against percentage inhibition (y-axis).
Percent inhibition was obtained through the formula:

%Inh = (Ao – Ac/Ao) x 100%

Where Ao is the absorbance of DPPH at the negative control (no antioxidant) and Ac is the
absorbance of DPPH with antioxidant.

Based from the data gathered, there was a decrease in absorbance values as the
amount of ascorbic acid given was increased. Upon computation, it was observed that a
decrease in absorbance value gives a higher percent inhibition (See Table 1 and Figure 1). A
color transition from violet to yellow was seen during the experiment, indicating the reduction of
the DPPH reagent, as the free radical scavenging takes place.

Table 1. Table of percent inhibition of ascorbic acid

Percent Inhibition (%) Concentration of Ascorbic acid
(mM)

91.45 0.300

53.60 0.150

47.44 0.105

39.51 0.060

19.2 0.030
 Figure 1. Percent inhibition of ascorbic acid

Same trend was exhibited by the antioxidant activity of -tocopherol. An increase in the
antioxidant concentration gave decreasing absorbance values, thus an increasing percent
inhibition (See Table 2). A violet to yellow color transition was also observed. The non-linearity
of the graph (See Figure 2) may be due to the wrong preparation for the 1.0 mM and 1.5 mM
concentration of -tocopherol.

Table 2. Table of percent inhibition of-tocopherol

Percent Concentration of a-tocopherol
inhibition (%) (mM)

27.54 2.0

19.23 1.5

26.81 1.0

13.75 0.5
 Figure 2. Percent inhibition of -tocopherol

In the DPPH assay, the vitamin C showed a more effective antioxidant activity than
vitamin E. We cannot compare the antioxidant activity of the two vitamins in terms of the IC50
values because the percent inhibition of -tocopherol did not reach 50%.

(TBARS DISCUSSION)

V. Conclusion

The DPPH Assay is used determine the free radical scavenging ability of antioxidants. A high
amount of antioxidant will cause a decrease in absorbance. High percent inhibition of Vitamin C
compared to Vitamin E shows that from these two antioxidants, it is the Vitamin C that works
using the process free radical scavenging.

The free radical scavenging activity of Vitamin C is done by reduction of ascorbate to ascorbate
free radical and dehydroascorbate.

(TBARS CONCLUSION)
VI. References

Alam, N., et al. (2013). Review on in vivo and in vitro methods evaluation of antioxidant activity.
Saudi Pharmaceutical Journal, 21(2): 143-152.

Best, B. (1990). General AntiOxidant Actions. Retrieved from:

http://www.benbest.com/nutrceut/AntiOxidants.html#conclude [Accessed 16 November
2014].

Molyneux, P. (2004). The use of the stable free radical diphenyl-picrylhydrazyl (DPPH) for
estimating antioxidant activity. Songklanakarin J. Sci. Technol., 26(2): 211-219.

Saha, M.R., et al. (2008). In vitro free radical scavenging activity of methanol extract of leaves
of Mimusops elengi Linn. Bangladesh Society for Veterinary Medicine, 6(2): 197-202.