The antioxidant potential of different plant extracts and pure compounds can be determined using numerous in vitro assays. Different assays evaluate the antioxidant activity of putative antioxidants based on different mechanisms, such as the ability to scavenge free radicals, the inhibition of lipid peroxidation, or the reductive potential to reduce Fe3+ to Fe2+. Each of these assays differs in their principles, characteristics, and applications.
There are number of different antioxidant in biological sample makes it difficult to measure each antioxidant separately. Therefore, an approach with multiple assays is highly advisable in evaluation of antioxidant capacity of biologic samples to measure various oxidation products. A single method is not recommended for the evaluation of the antioxidant activities of different plant products due to their complex composition (Chu, Chang, & Hsu, 2000; Nuutila et al., 2003). It has been reported that there is no simple universal method by which antioxidant activity can be measured accurately and quantitatively (Prior et al., 2005). Therefore, in the present study, the antioxidant potentials of Allium sativum and Zingiber officinale were evaluated using two different assays, namely 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and haemolysate catalytic assay.
5.2.1 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay
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DPPH radical scavenging assay is a simple and standard assay in antioxidant activity studies which allows rapid analysis of the radical scavenging activity of bioactive compounds or plant extracts (Amarowicz et al., 2004). The DPPH assay is sensitive in detecting natural compounds at low concentrations (Russo et al., 2005) and is independent of sample polarity. DPPH radical is one of the most used synthetic radicals for evaluation of antioxidant activity due to its stability in radical form and unaffected by certain side reactions, such as metalââ‚¬"ion chelation and enzyme inhibition.
The DPPH assay evaluates the ability of the antioxidants to neutralize DPPH free radicals by direct reduction through electron transfer or by radical quenching through hydrogen atom transfer. DPPH free radical is purple-coloured and possesses a characteristic absorption at the wavelength of 517 nm. Upon exposure to radical-scavengers, DPPH radical is reduced to stable yellow-coloured 1,1-diphenyl-2-picrylhydrazine (DPPH-H). The decrease in the DPPH radical absorbance at 517nm induced by antioxidant is taken as a measure of radical scavenging activity. A lower absorbance at 517 nm indicates a higher radical scavenging activity of the antioxidant or extract.
In the present study, the radical scavenging activity of the tested materials (pure antioxidants and plant extracts) was assessed on the bases of their IC50 values, which defined as the concentration of test material needed to scavenge 50% of DPPH radical present in the test solution. Lower IC50 value reflects better DPPH radical scavenging activity (Molyneux, 2004).
In this study, the DPPH radical scavenging activities of the extracts of Zingiber officinale were significantly higher than that of the extracts of Allium sativum. While in comparison to the ascorbic acid as standard, the extracts of Z. officinale exhibited slightly weaker radical scavenging effect.
All the extracts of Allium sativum exhibited very weak antioxidant activities with the DPPH radical scavenging activity of below 50% at all concentration tested. IC50 value was unable to be determined. The results obtained in this study are not in agreement with the earlier published data reported by Miller et al., (2000), which indicated that garlic is very high in antioxidants. The contradictory results might be partially due to the polarity of the extraction medium used, as 50% methanol was used for extraction by Miller and co-workers in their study while 100% methanol was used in this study (Nuutila et al., 2003). It has been shown that a wide variety of potential antioxidant compounds, such as flavonoids, phenolic acids and sulphur compounds, are present in the plants. Hence, extraction medium of different polarity can result in the extraction of different antioxidant compounds, which consequently leads to a wide variation in the antioxidant activity of the garlic.
According to Bozin et al., 2008, the extracts from mature garlic bulbs expressed significantly lower DPPH radical scavenging capacity than those obtained from the immature garlic plants. All the extracts were obtained from mature garlic bulbs in this study and thus, in agreement with the findings above. The DPPH radical scavenging activity was also reported to be dose-dependent (Bozin et al., 2008). This suggests that the concentrations of garlic extracts tested in the present study might be too low for the determination of IC50 values from the dose-response curve.
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The low radical scavenging activity of A. sativum could be explained partially by the low content of total phenolics and flavonoids in mature garlic bulb (Bozin et al., 2008).
The high antioxidant activity of Alliums and especially high RAS of garlic were reported by numerous investigators (Velioglu et al., 1998; Yin and Cheng, 1998; Miller et al., 2000).
However, RAS activity depended on both phenolics and sufur compounds of Alliums. On the other hand, Nuutila et al. (2003) reported that the lowest antioxidant activity was detected in garlic.
Among all the Z. officinale extracts tested, the crude chloroform extract was the most effective DPPH radical scavenger, with the IC50 value of 0.075mg/ml, while the crude water extract exhibited the weakest scavenging effect, with the IC50 value of 0.59mg/ml. Solvent systems used for extraction play a vital role in determining the type of phytochemicals being extracted, as non-polar solvents will extract non-polar substances while polar substances will be extracted by polar solvents. The highest scavenging activity in crude chloroform extract suggests that there might be compounds with medium polarity such as alkaloids, volatile oils, and aglycones in the crude extract that contributed to the high scavenging effect on DPPH radical.
In the present study, overall results indicated that all the Z. officinale extracts exhibited high radical scavenging activities. The high scavenging effect observed may depend greatly on the chemical compounds that exist in the plant. The presence of phenolic compounds, such as eugenol, shogaols, zingerone, gingerdiols, gingerols, diacetoxy--gingerdiol, in the ginger may contribute directly to its antioxidant action (Singh et al., 2008). Gingerol, the pungent factor in ginger, has been reported to exhibit strong in vitro antioxidant activity by Kikusaki and Nakatani (1993).
The important role of phenolic group in antioxidant activity has been reported and emphasized in many previous studies (Huang and Frankel, 1997; Baratta et al., 1998 and Singh et al., 2005). According to Silva et al. (2000), phenolic compounds exhibit significant scavenging effects against the DPPH free radical. The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet ant triplet oxygen or decomposing peroxides.
Ginger has been reported to demonstrate an antioxidant activity of comparatively as effective as that of ascorbic acid (Ahmed et al., 2000). The ability of the ginger extract to control the quantity of free radicals and significantly lower the induced lipid peroxidation has contributed to its high antioxidant effect (Basaga et al., 1997). Lipid peroxidation in tissues results in the production and propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids (PUFAs). This has been implicated in the pathogenesis of numerous diseases including atherosclerosis, diabetes, cancer and rheumatoid arthritis as well as in drug associated toxicity and aging (Halliwell & Gutteridge, 1989). Scavenging of free radical by dietary antioxidant, in addition to endogenous enzymes, is important to reduce the oxidative stress and consequently the oxidative damage on biological macromolecules in the body. Thus, dietary intake of ginger as natural antioxidant plays an important role in preventing or minimizing the oxidative damage caused by free radical in human body.
In recent years, there is a strong interest in the study of natural compounds with free radical scavenging capacity and many studies have been carried out on herbs and spices as natural antioxidant. Ginger, which has been used as a spice for centuries, has been indicated to possess potent antioxidant effect and is considered as a good source of antioxidant in many studies. According to Afshari et al., (2006), ginger can cause a decrease in lipid peroxidation, an increase of plasma antioxidant capacity and a reduction in renal nephropathy. It has also been suggested that ginger interferes with inflammatory processes.