Antioxidant And Free Radical Scavenging Activity Of Sorghum Bicolor (l.) Moench

Research Article ISSN: 0974-6943

Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(10),1659-1662

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, because 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 products. Free radicals are defined as molecules having an unpaired electron in the outer orbit and they are produced in the body, primarily as a result of aerobic metabolism. They are generally unstable and very reactive. The role of free radicals has been implicated in the causation of several diseases such as liver cirrhosis, atherosclerosis, cancer, aging, arthritis, diabetes 8 and the compounds that scavenge free radicals have great potential in ameliorating these disease processes 9. Sometimes these protective mechanisms are found not to be sufficient when compared to the insult produced to the body, hence the search for exogenous antioxidants is continued. Over the past three decades, the free radical theory has greatly stimulated interest in the role of dietary antioxidants in preventing many human diseases such as cancer, atherosclerosis, stroke, rheumatoid arthritis, neuro degeneration and diabetes.

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 activities. 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 Sorghum plants were grown in department of botany farm located at Andhra University, Visakhapaynam. Riboflavin was purchased from Loba chemie Pvt Ltd. (Bombay, India). Deoxiribose, Nitroblue tetrazolium (NBT) were purchased from Sisco Research Laboratories Pvt Ltd. (Mumbai, India). All other chemicals and reagents used were of analytical grade. Preparation of Extracts

For the preparation of acetone extracts, Sorghum seed powdered material of 1 kg was extracted with aqueous acetone (70%) by soxhlation. The crude extract was evaporated to dryness in a rotary film evaporator to give a yield of 210.11g (sorghum). For the assessment of free radical scavenging activities, the aqueous acetone extract 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 determined by the method of McCord and Fridovich 11. Which depends on *Corresponding author. light induced super oxide generation by riboflavin and the correspondDr. Varaprasad Bobbarala Scientist In-Charge, ing reduction of Nitroblue tetrazolium (NBT). The assay mixture conFor U Biosciences/IMMA Labs, tained the different concentrations of the extracts and EDTA (6µM A/4A, Park lane Residency, East point colony, Visakhapatnam, A.P-530017, India. containing 3µM NaCN), NBT (50µM), riboflavin (2µM) and phosTel.: + 91-9949129539 E-mail:[email protected]

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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 oxyControl OD gen species and free radicals, which play an important role the genesis 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. Determination of lipid peroxidation inhibiting activity:

Superoxide anion plays an important role in the formation of more reactive oxygen species such as hydrogen peroxide, hydroxyl radical, and singlet oxygen, which induce oxidative damage in lipids, proteins, and DNA. Therefore studying the scavenging activity of plant extract on superoxide radical is one of the most important ways of clarifying the mechanism of antioxidant activity. The concentration dependent percent inhibition of superoxide radical activity by acetone extract of sorghum bicolor and ascorbic acid was given in table 1 and shown in figure1.

Inhibition of lipid peroxidation was determined by the thiobarbituric acid method 12. Reaction mixture (0.5 ml) containing rat liver homogenate (0.1ml, 25% w/v) in tris-HCl buffer (40mM, pH-7). KCl (30mM), Ascorbic acid (0.06mM) and ferrous ion (0.16mM) and various concentrations of the extracts were incubated for one hour at 37ºC. The reaction mixture (0.4ml) was treated with sodium dodecyl 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 radi4ml 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 butanol-pyridine mixture (15: 1v/v) was added. Following vigorous shakIS2746 ing, the tubes were centrifuged and the absorbance of the organic 100 IS3040 layer containing the chromophore was read at 532nm. The percentage IS8887 90 IS33095 inhibition of lipid peroxidation by the extract was determined by comAscorbicacid 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 studying the competition between deoxyribose and the extracts for hydroxyl radicals generated from the Fe2+ / EDTA/H2O2 system (Fenton reaction). The hydroxyl radicals attack deoxy ribose which eventually results in thiobarbituric acid reacting substances (TBARS) formation 13. The reaction mixture contains deoxyribose (2.8 mM), ferrous sulphate (10mM). EDTA (10mM) H2O2 (1.0mM), phosphate buffer (0.1 M, pH 7.4) and various dilutions of the extracts. The reaction was incubated for 4 hours at 37ºC. Deoxy ribose degradation was mea-

40 30 20 10 0 50

100

200

300

400

500

Concentrations

Table-1: In vitro concentration dependent inhibition of superoxide radical by acetone extract of Sorghum bicolor/ascorbic acid Extracts/ Compound IS 2746 IS 3040 IS 33095 IS 8887 Ascorbic acid

Percentage 50 52.77±21.03 38.88±10.01 35.18±4.03 32.21±15.99 9.31±0.21

inhibition of Superoxide Radical. Concentration of extract/Ascorbic acid in micrograms (µg) 100 200 300 400 500 54.59±4.48 61.10±2.77 67.77±24.23 70.36±40.62 79.64±6.62 42.21±5.78 48.14±12.14 49.07±17.29 49.52±10.30 52.77±18.90 50.09±11.37 51.45±9.73 56.47±18.07 63.88±15.29 74.99±12.01 42.58±18.23 43.51±19.26 53.70±6.67 66.81±14.26 87.03±8.83 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 IS2746 IS3040 IS8887 IS33095 Ascorbic acid

90 80 70

IS 2746 IS 3040 IS 8887 IS 33095 Ascorbic acid

80 70 60 50

60 50

40

40

30

30

20

20

10

10

0 50

100

200

0 50

100

200

300

300

400

500

Concentrations

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 decomposition of lipid hydro peroxides. A single hydroxyl radical can result in formation of many molecules of lipid hydro peroxides in cell membrane, which may severely, disrupts its function, and lead to cell death 14,15. The concentration dependent percent inhibition of hydroxyl radical activity by acetone extract of sorghum bicolor and ascorbic 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 focused 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

IS 2746 IS 3040 IS 33095 IS 8887

100

43.37±6.96 47.05±5.56 22.02±5.39 26.75±2.94 7.79±.2.39 16.03±4.40 27.82±17.77 42.21±12.16

Ascorbic acid 20.46±3.70 37.01±0.83

200

300

400

500

51.97±0.53 29.75±5.66 18.16±5.44 61.73±3.05

54.97±2.04 37.58±3.35 25.79±4.35 62.50±4.02

63.08±2.34 52.04±1.20 37.86±21.82 69.26±13.55

71.04±3.38 61.92±0.62 55.35±3.39 72.30±15.32

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 inhibition 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 peroxides 79.22% and 72.30%. (Table 1, 2, 3) Hence, we concluded that pigmented varieties are showing a high antioxidant activity. REFERENCES 1. 2.

3. 4.

5.

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.2.Issue 10.October 2009

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