You are on page 1of 6

Experiment 4: antioxidant activity of common spices

Introduction
Aromatic and medicinal plants produce a diverse variety of natural products such as
aromatic compounds derived from the shikimate pathway, terpenes, terpenoids, alkaloids,
peptides, flavonoids, polyketides and fatty acids. Parts of such plants such as seeds, bark, leaves,
roots and stems are used in cooking.

Active principles of spices such as curcumin (turmeric), capsaicin (red chillies),eugenol


(cloves), linalool (coriander), piperine (black pepper), zingerone (zinger) and cuminaldehyde
(cumin) are reported to inhibit lipd peroxidation. They confer benefits to the food to which they
are added, e.g. aroma, taste, odour as well as antimicrobial, antioxidant and anticancer activities.

The antioxidant activity of plant compounds reside in their chemical structures, in which
resonance-stabilized functional groups such as phenolic groups are capable of scavenging free
radicals, thereby reducing, mitigating or abrogating the adverse effects that may be caused by
free radicals, e.g. reaction of free radicals with nucleic acids and proteins. Some products of free
radical-catalysed reactions may have negative effects on the cell or on the organism, including
interference with metabolic pathways, cell death, mutation and cancer.

In this experiment, you will prepare methanolic extracts of some spices that are very
commonly used in the kitchen: powdered coriander seeds, turmeric powder, chilli powder, white
pepper powder, mustard seedsand cumin, and quantify, then compare, the antioxidant activity of
these extracts.

A very convenient, and rapid, method to measure antioxidant activity is to use 2,2-diphenyl1,picrylhydrazyl (DPPH). This compound is a stable radical, purple in colour, and is converted to
a yellow colour by radical scavengers.
Figure 1: The reaction of DPPH, itself a free radical, with another free radical results in a colour
change from purple to yellow.

Apparatus
UV-visible spectrophotometer, 250 ml round bottom flask, condensor, heating mantle, rotary
evaporator, blender, micropipettes and tips (for volumes between 50 microlitres to 1ml), 10 ml
volumetric flasks (9 for each group), vortex
Materials and reagents
Aluminium foil, whatman 101 (Fast) filter paper, diameter 15cm, coriander seed powder,
turmeric powder, cumin, cloves, methanol, 0.6mM DPPH in methanol (to be made fresh, and
kept in the dark before use), quercetin standard, 50 ml of 1mg/ml in methanol, anhydrous
magnesium sulphate
All spices except cumin and cloves are supplied in powder form. You would need to subject only
cumin and cloves to blending in order to reduce these to powder form in order to improve the
efficacy of the extraction process.
Methods
5g of the powdered spice was weighed and put it in a 250 ml round bottom flask. 100 ml of
methanol was added and reflux for 1 hour. 5g of anhydrous magnesium sulphate was added to
adsorb traces of water from the samples, then filter the extract through a Whatman filter paper.
The extract subjected to rotary evaporation (weigh the round bottom flask used beforehand).
When all the methanol has been removed, extract was weighed. 100 ml of the extract in
methanol were prepared at a concentration of 1mg/ml.
The stock solution (1mg/ml) of each extract is to be diluted with methanol to obtain
concentrations of 0 (this is the control, which contains only methanol, and no extract) 5, 50, 100,
150,200, 250, 300, 400 and 500 micrograms/ml. 2ml of the extract pipette at various
concentrations into a test tube, and to each tube add 1 ml of 0.3mM DPPH solution. Each test
tube was shaked and stored in dark place for 30 minutes.
Absorbance of the contents of each tube at 517 nm were recorded. Lower absorbance values of
the reaction mixtures are indicative of higher radical scavenging activity. The capacity to
scavenge the DPPH radical is calculated using the following equation:

% DPPH scavenging effect = [A0-A1/A0] X 100


Where A0 is the absorbance of the control, and A1 the absorbance of the sample.

Plot a graph of % scavenging versus concentration, and determine the IC50 concentration (i.e. the
concentration that results in 50% scavenging).
The radical scavenging effect may be expressed in ascorbic acid equivalent antioxidant capacity
(AEAC), mgAA/100g.
AEAC sample (mgAA/100g) = IC50 (ascorbic acid)/IC50 (sample) X 105
Where IC50 (ascorbic acid)= 0.00387/mg/ml

Results
Cinnamon
concentration
(g/mL)
0
50
100
150
200
250
300
400
500

Absorbance
4.091
3.283
2.777
2.120
2.002
0.456
0.217
0.203
0.204

Turmeric
concentration
(g/mL)
0
50
100
150
200
250
300
400
500

Absorbance
4.000
3.436
3.215
3.068
2.914
2.270
1.420
1.030
0.620

Cloves

% DPPH
scavenging
19.751
32.119
48.179
51.063
88.854
94.696
95.038
95.013

% DPPH
scavenging
14.100
19.625
23.300
27.150
43.250
64.500
74.250
84.500

Cinnamon

Cloves
concentration
(g/mL)
0
50
100
150
200
250
300
400
500

Turmeric

Absorbance
4.000
3.135
2.005
1.676
1.174
0.734
0.675
0.412
0.218

Weight

4.9900g

5.008g

5.0021g

round 109.5372g

116.5118g

104.8513g

or
round 111.2483g
flask and

117.4927g

105.2718g

Weight
of
bottom flask:
Weight
bottom
product

Weight of product

1.7111g

0.9809g

0.4205g

Percentage yield

34.29%

19.59%

8.41%

The graph above is used to obtain the antioxidant capacity by using the equation as shown below.
AEAC sample (mgAA/100g) = IC50 (ascorbic acid)/IC50 (sample) X 105

Type of Spices
Cloves

IC50 (sample),(g/ml)
100

Antioxidant capacity (AEAC), mgAA/100g, 106


3.87

Turmeric

260

1.49

Cinnamon

200

1.94

IC50 (ascorbic acid) = 0.00387 mg/ml


Cloves,
IC50 = 100 g/ml = 0.100 mg/ml
Ascorbic acid equivalent antioxidant capacity (AEAC)
= IC50 (ascorbic acid)/ IC50 (sample) 105
= 0.00387 / 0.100 105
= 3870 mg AA/100g

Discussion
Radical scavenging ability tests aim to simulate basic mechanisms involved in lipid
oxidation by measuring either the reduction of stable radicals or radicals generated by radiolysis,
photolysis, or other reactions.

Cinnamon exhibited a second higher percentage of inhibition of oxidation than the other
spices analyzed. Cinnamon was a better superoxide radical scavenger than the other analyzed
spices and additives. The observation that the more polar antioxidants are more active in pure
lipids, and non-polar antioxidants most active in a polar substrate (e.g., oil-in-water emulsion),
and for which the term polar paradox has been introduced, may at least partially explain the
variation of antioxidative activity for different herbs and spices in different foods (Suhaj, 2006).
In previous studies showed that clove (in the Myrtaceae) had a very strong antioxidant
activity and a high level of phenolics The various antioxidant mechanisms of clove bud extracts
were attributed to a strong hydrogen-donating ability, a metal chelating ability, and their
effectiveness as good scavengers of hydrogen peroxide, superoxide, and free radicals (Corke, et
al., 2005). Our results showed that the clove bud extract was the most powerful phenolic
antioxidant and exhibited the strongest radical scavenging activity among the 3 spices.
Turmeric is a very stable spice and do not show a significant changes of its absorbance
and therefore exhibited a low IC50 inhibition of 1382.. Cloves, cinnamon and turmeric should be
planted in the same soil and environment so their total phenolic content and antioxidant capacity
can be measured more accurately.
Conclusion

IC50 inhibition concentration of cloves (3870 mg AA/100g) followed by cinnamon (2419 mg


AA/100g) and turmeric (1382 mg AA/100g).
References
Suhaj, M., 2006. Spice antioxidants isolation and their antiradical activity: a review. Journal of
Food Composition and Analysis, Volume 19, pp. 531-537.
Corke, H., Sun, M., Cai, Y.Z. and Shan, B., 2005. Antioxidant capacity of 26 spice extract and
characterization of their phenolic constituents. Journal of Agricultural and Food Chemistry,
Volume 53, pp. 7749-7759.