Assessment of Antioxidant Activity of Eugenol In Vitro and In Vivo
IC 50 values for the free radical scavengers in lipid peroxida- peroxida-tion system
3.3. Results and Conclusions
176 Nagababu et al.
6. Calculate the CYP450 concentration based on the difference in absorbance at 450 and 490 nm of reduced P450 with CO (as CO complex) and without CO using extinction coeffi-cient of 91 mM–1cm–1(Table 10.2).
3.2.5. G6Pase G6Pase activity (21) can be determined based on the release of inorganic phosphate according to Chen et al. (22).
1. Mix 50 L microsomes from the stock of 2 mg mL–1 with 850L 0.05 M maleic acid buffer, pH 6.5.
2. Add 100L substrate solution and mix well.
3. Incubate the mixture at 37◦C for 15 min.
4. Terminate the reaction by adding 1 mL of 10% (w/v) TCA and chill on ice.
5. Centrifuge the sample at 3,000 rpm for 15 min.
6. An aliquot of the supernatant can be used for determination of inorganic phosphate (Table 10.2).
3.2.6. Histopathological Examination of Liver
Fix small portions of liver sample (ca. 20 mm length × 15 mm width× 2–3 mm thickness) isolated from the middle lobe in 10%
neutral formalin. Process the tissue in an automated tissue proces-sor in ascending grades of isopropanol (70, 80, and 100%) for a total duration of 6–7 h at the rate of 1 h in each station, followed by 1 h each of three changes of chloroform for clearing, and finally 3 h each of two changes of paraffin infiltration (melting point of paraffin 58–60◦C), the second change being under vacuum.
Prepare paraffin blocks of the tissue and make sections (thick-ness 6 m) in a rotary manual or automated microtome using disposable knife blades. Stain the paraffin sections with Meyer’s;
hematoxylin–eosin (AFIP methods) and examine under ×10 objective with a final magnification of ×125. Grade hepatocel-lular necrosis, if any, as follows: no necrosis= 0; necrosis around centrilobular vein= 1.0; necrosis or fatty changes involving 1/3 of the lobule = 2.0; necrosis of more than 1/3 of lobule = 3.0 (see Fig. 10.4) and (Table 10.2).
3.3. Results and
Evaluation of Antioxidant Activity of Eugenol 177
Fig. 10.4. Effect of eugenol administration on CCl4-induced liver necrosis. (a) Photograph of liver from control rat, which received only vehicles (starch and peanut oil) exhibited normal liver architecture. (b) Histology of liver from CCl4-treated rat indicating centrizonal necrosis (necrosis score 2.0.) (c) Liver necrosis of rat treated with eugenol (1 mg kg–1body wt) + CCl4(necrosis score 1.5). (d) Liver necrosis showing moderate centrizonal necrosis from a rat, which received eugenol (5 mg kg–1body wt) + CCl4(necrosis score 1.25).
investigated. Mitochondria are the major sources for free radical generation, rich in polyunsaturated fatty acids, and highly suscep-tible to lipid peroxidation. Therefore, liver mitochondria are used as substrate for lipid peroxidation studies.
The results show that eugenol inhibits iron and•OH radical initiated lipid peroxidation with IC50 values of 10 and 14 M, respectively. The inhibitory activity of eugenol is five times more than ␣-tocopherol and 10 times less than BHT (Table 10.1).
Eugenol incorporates into mitochondrial membrane and micro-somal membrane inhibits lipid peroxidation by acting as chain-breaking agent (24, 25) (see Fig. 10.3).
Eugenol (0.2, 1.0, 5.0, or 25 mg kg–1 body wt) when given orally at three different times in relation to the time of CCl4 dos-ing (i.p administration of 0.4 mg kg–1 body wt), i.e., prior to (–1 h), along with (0 h), or after (+ 3 h), prevented significantly the rise in SGOT activity, lipid peroxidation, as well as liver necro-sis (see Fig. 10.4) (Table 10.2). The protective effect is more evident at 1 and 5 mg eugenol doses than that of 0.2 and 25 mg doses. However, the decrease in microsomal G6Pase activity and CYP450 content by CCl4treatment is not prevented by eugenol, suggesting that the damage to endoplasmic reticulum is not pro-tected (26) (Table 10.2). The protective effect of eugenol against
178 Nagababu et al.
CCl4-induced hepatotoxicity is due to interception of secondary radicals derived from oxidized lipids of endoplasmic reticulum rather than interference with generation and reactions of primary radicals (•CCl3/CClOO•).
4. Notes
1. Always use double-distilled water or ultra pure water with resistivity of≥18.2 M cm for preparation of all reagents.
2. Metal contamination is common in water and buffer solu-tions. Metals like iron and copper, in presence of reduc-ing agents, activate oxygen which oxidizes lipids, proteins, and DNA. Therefore, it is very important to use metal-free water for the preparation of reagents. Metal-free buffers and water can be prepared by passing through Chelex 100 (200–400 mesh, sodium form) resin.
3. Metal contamination in solutions can be tested by adding ascorbic acid to test solutions and monitor the change in absorbance at 265 nm. The loss of absorbance is an indica-tor of metal contamination. Metals promote autoxidation of ascorbic acid (27).
4. Sucrose could interfere in the estimation of malondialde-hyde. Avoid using sucrose media for preparation of mito-chondria or microsomes.
5. When preparing mitochondria or microsomes, keep liver on ice and homogenize in ice-cold saline to minimize the oxidation of lipids. To determine TBARS in whole tissue, keep the tissue piece in ice-cold TCA solution and homog-enize in same media.
6. Store microsomes and mitochondria in deaerated isotonic solutions in the refrigerator. Use within 72 h. Do not freeze the mitochondria or microsomes when using them as sub-strates for lipid peroxidation.
7. Phenolic antioxidants, vitamin E, and BHT are soluble in ethanol. Ethanol also scavenges radical species. Care should be taken to ensure that the total ethanol concentration in incubation mixture is less than 0.5%.
8. Prepare ferrous sulfate solution in water just before use.
Do not prepare iron solution in buffers. Always use freshly prepared solutions.
9. Add test compounds (antioxidant) 2–5 min prior to addi-tion of peroxidaaddi-tion inducers to substrates and mix it well.
Evaluation of Antioxidant Activity of Eugenol 179
10. Break-down products of lipid hydroperoxides formed dur-ing the heatdur-ing process also produce color similar to MDA.
Add 50L of 0.2% BHT to minimize these lipid hydroper-oxides contribution to MDA color soon after the reaction is completed.
11. Keep marbles on test tubes to minimize the evaporation of water while heating the reaction mixtures for MDA deter-mination. Replace if there is any water loss.
12. Stock hydrogen peroxide is standardized with potassium permanganate method or by molar extinction coefficient.
Working standard solutions of lower concentrations should be prepared just before use.
13. In some instances, antioxidants can interfere with the TBA reaction and suppresses the color development. This pos-sibility can be tested by adding a test compound to MDA standard or sample control soon after stopping the reaction with TCA.
Acknowledgments
This research was supported by National Institute of Nutri-tion, Indian Council of Medical Research, Hyderabad, India, and Intramural Research Program of the NIH, National Institute on Aging, USA.
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