Name of student : Wabatagore v

Student number : 201515533

Practical number : 5
Course : biochemistry 313
Due date : 20 /03 / 2017
TITLE : Spectrophotometric quantitation of antioxidant capacity of a plant leaf
extract.
ABSTRACT
Tocopherol is an antioxidant which prevents oxidation. These antioxidant are
found both in plants and humans. Free radicals react with oxygen atoms
damaging chemical reactions hence antioxidant are vital since they stop these
reactions. The phosphomolybdenum method is routinely applied to evaluate the
total antioxidant capacity of plant extracts. The amount of antioxidant in a leaf
can be determined spectrophotometrically through the use of standards.

INTRODUCTION
An antioxidant is as substance that is added to food and other products to
prevent harmful chemical reactions in which oxygen is combined with other
substances. Antioxidants help fight oxidation, a normal chemical process that
takes place in the body every day. It can be accelerated by stress, cigarette
smoking, and alcohol and these antioxidant are also found in plants. When there
are disruptions in the natural oxidation process, highly unstable and potentially
damaging molecules called free radicals are created (Mallet et al.,1994).
These free radicals are atoms or groups of atoms that contain an odd number of
electrons. They can be formed when certain molecules interact with oxygen.
Once formed, free radicals can start a chain of damaging chemical reactions.
Free radicals are not only generated by the body, they are present in foods you
eat as well as in the air you breathe (Hussain et al.,2013). Antioxidants can
safely interact with free radicals and stop the chain of damaging reactions
before damage is done to cells. There are several enzyme systems in the body
that scavenge free radicals, but we can also gain these helpful molecules from
foods that we eat. Some vitamins are antioxidants, such as vitamins C and E.
Some minerals are antioxidants, such as selenium and manganese, and there are
plant compounds that act as antioxidants such as beta carotene and lycopene .
Vitamin E, composed by α-, β-, γ-, and δ-tocopherols and tocotrienols, is
widespread in plant tissues including herbs, green leafy vegetables, leguminous
plants, oilseeds, grains, wheat, and to a lesser extent in animal foods such as
fish, eggs, milk, and liver (Daood et al.,1996).
The phosphomolybdenum method is routinely applied to evaluate the total
antioxidant capacity of plant extracts and to determine vitamin E in a variety of
grains and seeds, including corn and soya bean. The phosphomolybdenum
method, in combination with hexane monophasic extraction, has also been
adapted for the specific determination of vitamin E in seeds (Combs , 1992).

AIM
Is to measure the quantity of tocopherol in a leaf extract using a
spectrophotometer at 695nm and compare with standards to determine
tocopherol concentration in the leaf extract.

METHOD
Seven test tubes were labelled 1-7. Various solutions were thoroughly mixed as
follows:
Tube number 1 2 3 4 5 6 7
Phosphomolybdenum 5.0 5.0 5.0 5.0 5.0 5.0 5.0
reagent (ml)
Ethanol (ml) 0.5 0.4 0.3 0.2 0.1 0.0 0.0
Alpha-tocopherol 0.0 0.1 0.2 0.3 0.4 0.5 0.0
(ml)
Concebtration of 0.0 0.0 0.0 0.0 0.0 0.0 0.5
Leaf extract (ml)

This was done in duplicate. The test tubes were incubated at 80⁰C for 15
minutes. Afterwards, the tubes were cooled and the absorbance was read at
695nm.
RESULTS AND CALCULATIONS
Table 1.: absorbance of 10mM α-tochopherol and 5ml of phosphomolybdenum
solution in different test tubes with different volumes of α-tochopherol and one
tube with leaf extract at 695nm.
Tube Absorbance at 695nm
number
First sample Second sample Average
1 0.000 0.000 0.000
2 0.156 0.140 0.148
3 0.169 0.172 0.171
4 0.176 0.182 0.179
5 0.221 0.230 0.225
6 0.249 0.251 0.250
7 2.204 2.201 2.202

The concentrations of the solution of phosphomolybdenum α-tocopherol were

calculated as follows ,

C1V1 = C2V2 , where C1 is the initial concentration , C2 is the final concentration

and V1 is the initial volume , V2 is the final volume
For test the concentration was ;
10 (0.0 ml) = C2( 5.5ml)
C2 = 0.00000M
For test tube 2
10( 0.1ml) = C2(5.5ml)
C2 = 0.182mM
For test tube 3
10( 0.2ml) = C2(5.5ml)
C2 = 0.364mM
For test tube 4
10( 0.3ml) = C2(5.5ml)
C2 = 0.546mM
For test tube 5
10( 0.4ml) = C2(5.5ml)
C2 = 0.727mM
For test tube 6
10( 0.5ml) = C2(5.5ml)
C2 = 0.909mM

Table 2 : tabular representation of the calculated concentrations of

phosphomolybdenum solution with α- tocopherol in different test tubes

Tube number 1 2 3 4 5 6 7
Absorbance at 695nm 0.000 0.148 0.171 0.179 0.225 0.251 0.201
α-tocopherol solution 0.000 0.182 0.364 0.546 0.727 0.909 -
concentration (mM)
 0.3

y = 0.2349x + 0.0555
0.25

0.2
Absorbance (695nm)

0.15

0.1

0.05

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Concentration (Mm)

Figure 1 : graphically representation of absorbance against concentration

Concentration of unknown solution from the graph
y = 0.2349x + 0.0555
2.202 = 0.239x + 0.0555
X = 8.9mM (concentration of unknown).
DISCUSSION
Spectrophotometric method was used for the quantitative determination of
antioxidant capacity in the leaf extract by comparing it with the standards.
Ethanol was a solvent which was used in the extraction of α-tocopherol and
other lipid-soluble antioxidants. Test tube 1 was used as a blank as it was a
control for the rest of the test tubes which were under study (Mallet et al.,1994).
The assay was based on the reduction of phosphomolybedum (VI) to
phosphomolybedum (V) by the sample analyte and the subsequent formation of
a green phosphate/ phosphomolybedum (V) complex. The kinetics of the
formation of the phosphomolybdenum complex was monitored at 80 oC with α-
tocopherol as the reducing agent. In the formation of the complex ,
phosphomolybdenum was reduced by α-tocopherol and the α-tocopherol was
itself oxidised in the process. The formation of the complex was dependent on
the α-tocopherol concentration (Daood et al.,1996).
The experiment was done to optimize the formation and determination of the
green phosphomolybdenum complex with α-tocopherol as the reducing species.
The absorbance of the solution was read at 695nm since the
phosphomolybdenum complex had a characteristic maximum at 695 nm
(Hussain et al.,2013). After the test tubes were removed from the incubator a
green complex was observed. From test tube 2 to the last test tube the greener
colour became more darker with the increase in the concentration of α-
tocopherol . The absorbance of the α-tocopherol solution increased with its
increase in concentration (Combs , 1992).
According to the spectrophotometric results obtained , the absorbance values
increased with increase in the concentration of α-tocopherol , since formation of
the complex was dependent on the concentration of α-tocopherol. Through the
use of standards , their concentrations and absorbances which were measured at
695nm , the concentration of α-tocopherol present in the leaf extract was
determined (Hussain et al.,2013).
Since the absorbance value of the unknown concentration was found to be out
of range , as compared to those of standards , the concentration of the unknown
was determined from an excel graph . From the excel graph, the concentration
of α-tocopherol in the leaf extract was found to be 8.9mM. This concentration
was too concentrated hence its concentration was not in the standard ranges (
Hadi and Hayati 2006).
REFERENCE
Combs, G. F. (1992) The Vitamins: Fundamental Aspects in Nutrition and
Health, Academic Press, San Diego.
Daood, H. G., Vinkler, M., Markus, F., Hebshi, E. A., and Biacs, P. A. (1996)
Food Chem. 55, 365– 372.
Hadi A, Hayati N (2006) Chemical composition and activities of antioxidant
compounds in red pitaya fruit (Hylocereus sp.), and effects on glucose and lipid
profile level of induced hyperglycemia rats. Thesis, Universiti Putra Malaysia
Hussain A, Larsson H, Olsson M, Kuktaite R, Grausgruber H, Johansson E
(2012) Is organically produced wheat a source of tocopherols and tocotrienols
for health food. Food Chem 132:1789–1795
Hussain N, Jabeen Z, Li Y, Chen M, Li Z, Guo W, Shamsi I, Chen X, Jiang L
(2013) Detection of tocopherol in oilseed rape (Brassica napus L.) using gas
chromatography with flame ionization detector. J Integ Agr 12:803–814
Mallet, J. F., Cerrati, C., Ucciani, E., Gamisans, J., and Gruber, M. (1994) Food
Chem. 49, 61–65.