Varaprasad Bobbarala et al.

/ Journal of Pharmacy Research 2009, 2(10),1659-1662

Research Article
ISSN: 0974-6943 Available online through
www.jpronline.info
Antioxidant and free radical scavenging activity of Sorghum bicolor (L.) Moench
T. Bangaru Naidu 1, S. Nageswara Rao1, N. Sarada Mani 1, Varaprasad Bobbarala2*
1Department of Botany, Andhra University, Visakhapatnam-3,A.P, India

2For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam-17,A.P, India

Received on: 20-05-2009; Accepted on:15-07-2009

ABSTRACT
In the present study, the antioxidant properties of acetone extracts from four cultivated varieties of Sorghum bicolor i.e., IS 2746 and IS 8887,
pigmented varieties IS 3040 and IS 33095 are non pigmented varieties were estimated by three different methods. The percentage inhibition
of superoxide, hydroxyl and lipid peroxides was maximum at 50 to 500µg respectively, which is also supported by significant positive
correlation obtained from percentage of inhibition versus concentration used. Of the four genotypes used in this present study, IS-8887 has
shown maximum of 87.03% inhibition of superoxide radical, while the genotype IS-2746 has shown maximum inhibition in hydroxyl, lipids
peroxides 79.22% and 72.30% in this order.

Keywords: Sorghum bicolor; Antioxidant properties, Ascorbic acid Equivalents, Free radical scavengers.

INTRODUCTION
Sorghum bicolor is a major cereal food crop used in many
parts of the world. Sorghum contains high level of diverse phenolic
compounds that may provide health benefits. Phenolic compounds
are generally regarded as desirable compounds of human food, be-
cause of their antioxidant activity. High levels of poly flavanols 1, 2, 3
anthocyanins, phenolicacids 4,5, 6,7 and other antioxidant compounds
have been reported in sorghums. However, data are hard to find on
antioxidant activities of the specialty sorghum and / or their prod-
ucts.
peroxidase 10. As far as we know from previous literature, some of the
selected herbal drugs are known to possess superoxide, or hydroxyl,
or DPPH radical scavenging and lipid peroxidation inhibition activi-
ties. There is no detailed study on free radical scavenging activities
on sorghum. Hence, a detailed study was carried out on superoxide
radical, hydroxyl radical and lipid peroxidation inhibiting activities of
the acetone extract of sorghum.
MATERIALS AND METHODS

Free radicals are defined as molecules having an unpaired Sorghum plants were grown in department of botany farm
electron in the outer orbit and they are produced in the body, prima- located at Andhra University, Visakhapaynam. Riboflavin was pur-
rily as a result of aerobic metabolism. They are generally unstable and chased from Loba chemie Pvt Ltd. (Bombay, India). Deoxiribose,
very reactive. The role of free radicals has been implicated in the Nitroblue tetrazolium (NBT) were purchased from Sisco Research
causation of several diseases such as liver cirrhosis, atherosclerosis, Laboratories Pvt Ltd. (Mumbai, India). All other chemicals and re-
cancer, aging, arthritis, diabetes 8 and the compounds that scavenge agents used were of analytical grade.
free radicals have great potential in ameliorating these disease pro-
cesses 9. Sometimes these protective mechanisms are found not to be Preparation of Extracts
sufficient when compared to the insult produced to the body, hence
the search for exogenous antioxidants is continued. Over the past For the preparation of acetone extracts, Sorghum seed pow-
three decades, the free radical theory has greatly stimulated interest dered material of 1 kg was extracted with aqueous acetone (70%) by
in the role of dietary antioxidants in preventing many human diseases soxhlation. The crude extract was evaporated to dryness in a rotary
such as cancer, atherosclerosis, stroke, rheumatoid arthritis, neuro film evaporator to give a yield of 210.11g (sorghum). For the assess-
degeneration and diabetes. ment of free radical scavenging activities, the aqueous acetone ex-
tract were redissolved in water for assessment of in vitro free radical
Antioxidant actions might be exerted by inhibiting genera- scavenging activity.
tion of reactive oxygen species and reactive nitrogen species, or by
directly scavenging free radicals or by raising the levels of endog- Determination of superoxide scavenging activity:
enous antioxidant defenses, for example, by up regulating expression
of the genes encoding superoxide dismutase, catalase or glutathione Superoxide scavenging activity of the extracts was deter-
mined by the method of McCord and Fridovich 11. Which depends on
*Corresponding author.
Dr. Varaprasad Bobbarala light induced super oxide generation by riboflavin and the correspond-
Scientist In-Charge, ing reduction of Nitroblue tetrazolium (NBT). The assay mixture con-
For U Biosciences/IMMA Labs,
A/4A, Park lane Residency, East point colony, tained the different concentrations of the extracts and EDTA (6µM
Visakhapatnam, A.P-530017, India. containing 3µM NaCN), NBT (50µM), riboflavin (2µM) and phos-
Tel.: + 91-9949129539
E-mail:varaprasadphd@rediffmail.com
Journal of Pharmacy Research Vol.2.Issue 10.October 2009 1659-1662
 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(10),1659-1662
phate buffer (58mM, p H 7.8) to give a total volume of 3ml. the tubes sured as TBA reactive substances by the method of Ohkawa et al., 12
were uniformly illuminated for 15 minutes and there after the optical and the percentage of inhibition was calculated from the control where
density (OD) was measured at 560nm. The percentage inhibition by no test compound was added. The percentage inhibition of hydroxyl
the extracts of superoxide production was evaluated comparing the radicals by the extracts was determined by comparing the absorbance
absorbance values of control and experimental tubes. Percentage of values of the control and the experimental tubes as calculated for
inhibition by the extracts of superoxide production was calculated superoxide radical assay.
using the formula
RESULTS AND DISCUSSION
Control OD-Test OD
% inhibition = ————————— X 100 Recently much attention has been focused on reactive oxy-
Control OD gen species and free radicals, which play an important role the gen-
esis of various diseases. Hence, the present study is focused on the
The OD obtained with each concentration of the extracts determination of antioxidant activity by superoxide, hydroxyl and lipid
and ascorbic acids was plotted on a graph taking concentration on x peroxidation. Our results are compared with excellent stranded drug
axis and percentage inhibition on Y-axis, the graph was extrapolated like Ascorbic acid.
to find the concentration needed for 50% inhibition.
Superoxide anion plays an important role in the formation of
Determination of lipid peroxidation inhibiting activity: more reactive oxygen species such as hydrogen peroxide, hydroxyl
radical, and singlet oxygen, which induce oxidative damage in lipids,
Inhibition of lipid peroxidation was determined by the proteins, and DNA. Therefore studying the scavenging activity of
thiobarbituric acid method 12. Reaction mixture (0.5 ml) containing rat plant extract on superoxide radical is one of the most important ways
liver homogenate (0.1ml, 25% w/v) in tris-HCl buffer (40mM, pH-7). of clarifying the mechanism of antioxidant activity. The concentration
KCl (30mM), Ascorbic acid (0.06mM) and ferrous ion (0.16mM) and dependent percent inhibition of superoxide radical activity by ac-
various concentrations of the extracts were incubated for one hour at etone extract of sorghum bicolor and ascorbic acid was given in table
37ºC. The reaction mixture (0.4ml) was treated with sodium dodecyl 1 and shown in figure1.
sulphate (SDS-0.2ml 8.1%), thio barbiuric acid (TBA-1.5ml, 0.8%) and
acetic acid (1.5ml, 20pH-3.5). The total volume was then made up to
Fig 1: In vitro concentration dependent inhibition of superoxide radi-
4ml by adding distilled water and kept in oil bath at 100ºC for one hour.
cal by acetone extract of Sorghum bicolor and ascorbic acid
After the mixture had been cooled 1ml distilled water and 5ml of bu-
tanol-pyridine mixture (15: 1v/v) was added. Following vigorous shak-
ing, the tubes were centrifuged and the absorbance of the organic IS2746
100 IS3040
layer containing the chromophore was read at 532nm. The percentage IS8887
inhibition of lipid peroxidation by the extract was determined by com- 90 IS33095
Ascorbicacid
paring the absorbance values of the control and the experimental 80
tubes as calculated for superoxide radical assay.
70

60
Deoxy ribose degradation method:
50

Hydroxyl radical scavenging activity was measured by 40

studying the competition between deoxyribose and the extracts for
30
hydroxyl radicals generated from the Fe2+ / EDTA/H2O2 system (Fenton
reaction). The hydroxyl radicals attack deoxy ribose which eventually 20

results in thiobarbituric acid reacting substances (TBARS) formation 10

13. The reaction mixture contains deoxyribose (2.8 mM), ferrous sul-
0
phate (10mM). EDTA (10mM) H2O2 (1.0mM), phosphate buffer (0.1 50 100 200 300 400 500
M, pH 7.4) and various dilutions of the extracts. The reaction was Concentrations
incubated for 4 hours at 37ºC. Deoxy ribose degradation was mea-

Table-1: In vitro concentration dependent inhibition of superoxide radical by acetone extract of Sorghum bicolor/ascorbic acid
Extracts/ Compound Percentage inhibition of Superoxide Radical. Concentration of extract/Ascorbic acid in micrograms (µg)
50 100 200 300 400 500
IS 2746 52.77±21.03 54.59±4.48 61.10±2.77 67.77±24.23 70.36±40.62 79.64±6.62
IS 3040 38.88±10.01 42.21±5.78 48.14±12.14 49.07±17.29 49.52±10.30 52.77±18.90
IS 33095 35.18±4.03 50.09±11.37 51.45±9.73 56.47±18.07 63.88±15.29 74.99±12.01
IS 8887 32.21±15.99 42.58±18.23 43.51±19.26 53.70±6.67 66.81±14.26 87.03±8.83
Ascorbic acid 9.31±0.21 17.06±0.48 43.86±0.69 68.97±1.01 76.77±0.48 83.04±0.58

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 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(10),1659-1662
Table-2: In vitro concentration dependent inhibition of hydroxyl radical by acetone extract of sorghum bicolor/ascorbic acid
Extracts/ Compound Percentage inhibition of Hydroxyl radical .Concentration of extract/Ascorbic acid in micrograms (µg)
50 100 200 300 400
IS 2746 42.37±4.52 53.17±14.69 54.75±6.32 73.64 ±19.79 79.22±24.92
IS 3040 21.42±8.91 24.28±10.26 37.45±10.87 41.26±11.83 53.01±16.05
IS 8887 19.83±8.05 25.23±6.24 33.67±16.22 36.34±16.80 52.21±1.14
IS 33095 14.63±4.49 17.77±2.28 21.58±8.43 26.58±2.36 51.42±28.02
Ascorbic acid 5.22±1.52 15.47±1.00 69.12±2.68 74.47±0.58 77.74±0.57

Fig 2: In vitro concentration dependent inhibition of hydroxyl radi- Fig 3: In vitro concentration dependent inhibition of lipid peroxidation
cal by acetone extract of Sorghum bicolor and ascorbic acid radical by acetone extract of sorghum bicolor and ascorbic acid

IS 2746
IS2746 80 IS 3040
90 IS3040 IS 8887
IS8887 IS 33095
70
80 IS33095 Ascorbic acid
Ascorbic acid
60
70

50
60

50 40

40 30

30 20

20 10

10
0
50 100 200 300 400 500
0
Concentrations
50 100 200 300 400
Concentrations

Among the ROS, the hydroxyl radicals are the most reactive
and predominant radicals generated endogenously during aerobic
metabolism. Due to the high reactivity, the radicals have a very short
biological half-life. The generated hydroxyl radicals initiate the lipid
peroxidation process and /or propagate the chain process via decom-
position of lipid hydro peroxides. A single hydroxyl radical can result
in formation of many molecules of lipid hydro peroxides in cell mem-
brane, which may severely, disrupts its function, and lead to cell
death 14,15. The concentration dependent percent inhibition of hy-
droxyl radical activity by acetone extract of sorghum bicolor and ascor-
bic acid were given in table 2 and shown in figure 2.
Lipid peroxides:
Lipid peroxidation, which involves a series of free radical
meditated chain reaction processes, is also associated with several
types of biological damage. Therefore much attention has been fo-
cused on the use of natural antioxidants to inhibit lipid peroxidation
and to protect from damage due to free radicals 16. The concentration
dependent lipid peroxidation inhibition activity by acetone extract of
sorghum bicolor and ascorbic acid were given in table 3 and shown in
figure 3.

Table-3: In vitro concentration dependent inhibition of lipid peroxidation radical by Acetone extract of sorghum bicolor/ascorbic acid

Extracts/
Compound Percentage inhibition of Lipid peroxidation Radical Concentration of extract/Ascorbic acid in micrograms (µg)

50 100 200 300 400 500

IS 2746 43.37±6.96 47.05±5.56 51.97±0.53 54.97±2.04 63.08±2.34 71.04±3.38
IS 3040 22.02±5.39 26.75±2.94 29.75±5.66 37.58±3.35 52.04±1.20 61.92±0.62
IS 33095 7.79±.2.39 16.03±4.40 18.16±5.44 25.79±4.35 37.86±21.82 55.35±3.39
IS 8887 27.82±17.77 42.21±12.16 61.73±3.05 62.50±4.02 69.26±13.55 72.30±15.32
Ascorbic acid 20.46±3.70 37.01±0.83 51.65±0.58 59.00±0.35 68.97±1.38 74.33±1.73

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 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(10),1659-1662
The results of percentage inhibition of superoxide, hydroxyl, and lipid
peroxides increased with the increasing concentration of aqueous
acetone extracts of all four varieties and maximum percentage of inhi-
bition was observed at 50 to 500µg concentration respectively. Of the
different genotypes used in this present study IS 8887 has been shown
maximum of 87.03% inhibition of superoxide radical, while the variety
of IS 2746 has been shown maximum inhibition in hydroxyl, lipid per-
oxides 79.22% and 72.30%. (Table 1, 2, 3) Hence, we concluded that
pigmented varieties are showing a high antioxidant activity.
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