Kailash Prasad

Oxygen free radicals and hypercholesterolemic atherosclerosis: Effect of vitamin E☆

We investigated the effects of a high-cholesterol diet in the presence and absence of vitamin E on the lipid peroxidation product malondialdehyde of blood and aortic tissue, the oxygen-free-radical-producing activity of polymorphonuclear leukocytes (PMNs) (PMN chemiluminescence), and the blood lipid profile in rabbits. The animals were divided into four groups each of which comprised 10 rabbits. Rabbits in group I received a regular rabbit chow diet; those in group II received vitamin E; those in group III received high cholesterol + vitamin E; and those in group IV received a high-cholesterol diet. Blood concentrations of triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), malondialdehyde, and PMN chemiluminescence were measured. The aorta of each rabbit was removed at the end of the protocol for assessment of atherosclerotic changes (gross and microscopic) and malondialdehyde. Serum triglycerides, total cholesterol, HDL-C, LDL-C, and VLDL-C increased while HDL/LDL ratio decreased in groups III and IV but remained unchanged in group I. There was an increase in the HDL-C component and HDL/LDL ratio and a decrease in the LDL-C component and triglycerides in group II. Blood and aortic tissue malondialdehyde increased in group IV but decreased in groups II and III. PMN chemiluminescence increased in groups III and IV. Atherosclerotic changes were marked in group IV as compared with those in group III. However, histologic changes in the aortas were similar in groups III and IV. The increased levels of blood and aortic tissue malondialdehyde and PMN chemiluminescence, which were associated with development of atherosclerosis, suggest a role of oxygen free radicals in the pathogenesis of hypercholesterolemia-induced atherosclerosis. The protection afforded by vitamin E, which was associated with a decrease in blood and aortic tissue malondialdehyde concentration in spite of hypercholesterolemia, supports the hypothesis that oxygen free radicals are involved in the development of hypercholesterolemic atherosclerosis.

Lipid peroxidation and activity of antioxidant enzymes in diabetic rats

We hypothesized that oxygen free radicals (OFRs) may be involved in pathogenesis of diabetic complications. We therefore investigated the levels of lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS) and activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] in tissues and blood of streptozotocin (STZ)-induced diabetic rats. The animals were divided into two groups: control and diabetic. After 10 weeks (wks) of diabetes the animals were sacrificed and liver, heart, pancreas, kidney and blood were collected for measurement of various biochemical parameters. Diabetes was associated with a significant increase in TBARS in pancreas, heart and blood. The activity of CAT increased in liver, heart and blood but decreased in kidney. GSH-Px activity increased in pancreas and kidney while SOD activity increased in liver, heart and pancreas. Our findings suggest that oxidative stress occurs in diabetic state and that oxidative damage to tissues may be a contributory factor in complications associated with diabetes.

Reduction of Serum Cholesterol and Hypercholesterolemic Atherosclerosis in Rabbits by Secoisolariciresinol Diglucoside Isolated From Flaxseed

BACKGROUND Secoisolariciresinol diglucoside (SDG) is a plant lignan isolated from flaxseed. Lignans are platelet-activating factor-receptor antagonists that would inhibit the production of oxygen radicals by polymorphonuclear leukocytes. SDG is an antioxidant. Antioxidants studied thus far are known to reduce hypercholesterolemic atherosclerosis. The objective of this study was to determine the effect of SDG on various blood lipid and aortic tissue oxidative stress parameters and on the development of atherosclerosis in rabbits fed a high-cholesterol diet. METHODS AND RESULTS Rabbits were assigned to 4 groups: group 1, control; group 2, SDG control (15 mg. kg body wt-1. d-1 PO); group 3, 1% cholesterol diet; and group 4, same as group 3 but with added SDG (15 mg. kg body wt-1. d-1 PO). Blood samples were collected before (time 0) and after 4 and 8 weeks of experimental diets for measurement of serum triglycerides, total cholesterol (TC), and LDL, HDL, and VLDL cholesterol (LDL-C, HDL-C, and VLDL-C). The aorta was removed at the end of the protocol for assessment of atherosclerotic plaques; malondialdehyde, an aortic tissue lipid peroxidation product; and aortic tissue chemiluminescence, a marker for antioxidant reserve. Serum TC, LDL-C, and the ratios LDL-C/HDL-C and TC/HDL-C increased in groups 3 and 4 compared with time 0, the increase being smaller in group 4 than in group 3. Serum HDL-C decreased in group 3 and increased in group 4 compared with time 0, but changes were lower in group 3 than in group 4. SDG reduced TC and LDL-C by 33% and 35%, respectively, at week 8 but increased HDL-C significantly, by>140%, as early as week 4. It also decreased TC/LDL-C and LDL-C/HDL-C ratios by approximately 64%. There was an increase in aortic malondialdehyde and chemiluminescence in group 3, and they were lower in group 4 than in group 3. SDG reduced hypercholesterolemic atherosclerosis by 73%. CONCLUSIONS These results suggest that SDG reduced hypercholesterolemic atherosclerosis and that this effect was associated with a decrease in serum cholesterol, LDL-C, and lipid peroxidation product and an increase in HDL-C and antioxidant reserve.

Antioxidant activity of secoisolariciresinol diglucoside-derived metabolites, secoisolariciresinol, Enterodiol, and enterolactone

Secoisolariciresinol diglucoside (SDG), an anti-oxidant isolated from flaxseed, is metabolized to secoisolariciresinol (SECO), enterodiol (ED), and enterolactone (EL) in the body. The effectiveness of SDG in hypercholesterolemic atherosclerosis, diabetes, and endotoxic shock could be due to these metabolites. These metabolites may have antioxidant activity. However, the antioxidant activity of these metabolites is not known. The antioxidant activity of SECO, ED, and EL was investigated using chemiluminescence (CL) of zymosan-activated polymorphonuclear leukocytes (PMNLs) [PMNL-CL]. Other antioxidants (SDG and vitamin E) were also used for comparison. SDG, SECO, ED, EL, and vitamin E, each in the concentration of 0.5, 1.0, 2.5, 5.0 and 10.0 mg/ml, produced a concentration-dependent reduction in zymosan-activated PMNL-CL. SDG, SECO, ED, EL, and vitamin E, in the concentration of 2.5 mg/ml, produced a reduction of zymosan-activated PMNL-CL by 23.8%, 91.2%, 94.2%, 81.6% and 18.7%, respectively. Activated PMNLs produce reactive oxygen species and luminol-dependent CL reflects the amount of oxygen species generated from activated PMNLs. The reduction of PMNL-CL, therefore, reflects the antioxidant activity of the compounds studied. These results suggest that the metabolites of SDG have antioxidant activity. The antioxidant activity was highest with SECO and ED and lowest with vitamin E. The antioxidant potency of SECO, ED, EL, and SDG was 4.86, 5.02, 4.35, and 1.27 respectively, as compared to vitamin E. SECO, ED and EL are respectively 3.82, 3.95, and 3.43 more potent than SDG.

Hydroxyl radical-scavenging property of secoisolariciresinol diglucoside (SDG) isolated from flax-seed

Recently there has been a moderate resurgence in the use of flax-seed in a variety of ways including bread. The scientific basis of its use is very limited. There is some claim for beneficial effects in cancer and lupus nephritis. These claims could be due to its ability to scavenge oxygen radicals. However, its antioxidant activity is not known. Recently a method has been developed to isolate secoisolariciresinol diglucoside (SDG) from defatted flax-seed in large quantity (patent pending). We investigated the ability of SDG to scavenge úOH using high pressure liquid chromatography (HPLC) method. úOH was generated by photolysis of H2O2 (1.25-10.0 \sgmaelig;moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce úOH-adduct products 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA. H2O2 produced a concentration-dependent úOH as estimated by 2,3-DHBA and 2,5-DHBA. A standard curve was constructed for known concentrations of 2,3-DHBA and 2,5-DHBA against corresponding area under the peaks which then was used for measurement of 2,3-DHBA and 2,5-DHBA generated by UV irradiation of H2O2 in the presence of salicylic acid. SDG in the concentration range of 25, 50, 100, 250, 500, 750, 1000 and 2000 \sgmaelig;g/ml (36.4, 72.8, 145.6, 364.0, 728.0, 1092.0, 1456.0 and 2912.0 \sgmaelig;M respectively) produced a concentration-dependent decrease in the formation of 2,3-DHBA and 2,5-DHBA, the inhibition being 4 and 4.65% respectively with 25 \sgmaelig;g/ml (36.4 \sgmaelig;M) and 82 and 74% respectively with 2000 \sgmaelig;g/ml (2912.0 \sgmaelig;M). The decrease in úOH-adduct products was due to scavenging of úOH not and by scavenging of formed 2,3-DHBA and 2,5-DHBA. SDG prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner in the concentration range from 319.3-2554.4 \sgmaelig;M. These results suggest that SDG scavenges úOH and therefore has an antioxidant activity.

Antioxidant activity of allicin, an active principle in garlic

Garlic has been claimed to be effective against diseases, in the pathophysiology of which oxygen free radicals (OFRs) have been implicated. Effectiveness of garlic could be due to its ability to scavenge OFRs. However, its antioxidant activity is not known. We investigated the ability of allicin (active ingredient of garlic) contained in the commercial preparation Garlicin to scavenge hydroxyl radicals (·OH) using high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H2O2 (1.25–10 μmoles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce ·OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). H2O2 produced a concentration-dependent ·OH as estimated by ·OH adduct products 2,3-DHBA and 2,5-DHBA. Allicin equivalent in Garlicin (1.8, 3.6, 7.2, 14.4, 21.6, 28.8 and 36 μg) produced concentration-dependent decreases in the formation of 2,3-DHBA and 2,5-DHBA. The inhibition of formation of 2,3-DHBA and 2,5-DHBA with 1.8 μg/ml was 32.36% and 43.2% respectively while with 36.0 μg/ml the inhibition was approximately 94.0% and 90.0% respectively. The decrease in ·OH adduct products was due to scavenging of ·OH and not by scavenging of formed ·OH adduct products. Allicin prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner. These results suggest that allicin scavenges ·OH and Garlicin has antioxidant activity.

Dietary flax seed in prevention of hypercholesterolemic atherosclerosis

Oxygen free radicals (OFRs) have been implicated in the development of hypercholesterolemic atherosclerosis. Flax seed is the richest source of omega-3 fatty acid and lignans. omega-3 Fatty acid suppresses the production of interleukin-1 (IL-1), tumor necrosis factor (TNF) and leukotriene B4 (LTB4), and of OFRs by polymorphonuclear leukocytes (PMNLs) and monocytes. Lignans possess anti-platelet activating factor (PAF) activity and are antioxidant. PAF, IL-1, TNF and LTB4 are known to stimulate PMNLs to produce OFRs. Flaxseed would, therefore, reduce the levels of OFRs and hence would prevent the development of hypercholesterolemic atherosclerosis. The effects of dietary flax seed on a high cholesterol diet induced atherosclerosis, lipid profile and OFR-producing activity of PMNLs (PMNL-CL) were investigated in rabbits. The rabbits were divided into 4 groups: group I, control; group II, flax seed diet (7.5 g/kg daily, orally); group III, 1% cholesterol diet; and group IV, same as group III but received flax seed (7.5 g/kg daily, orally). Blood samples were collected before and after 4 and 8 weeks on their respective diets for biochemical measurements and aortae were removed at the end of 8 weeks for estimation of atherosclerotic changes. The high cholesterol diet increased the serum level of total cholesterol (TC) and PMNL-CL without altering the levels of serum triglycerides (TG). These changes were associated with a marked development of atherosclerosis in the aorta. Flax seed reduced the development of aortic atherosclerosis by 46% and reduced the PMNL-CL without significantly lowering the serum cholesterol. Flax seed in normocholesterolemic rabbits increased serum total cholesterol and decreased PMNL-CL without significantly affecting the serum TG. Modest dietary flax seed supplementation is effective in reducing hypercholesterolemic atherosclerosis markedly without lowering serum cholesterol. Its effectiveness against hypercholesterolemic atherosclerosis could be due to suppression of enhanced production of OFRs by PMNLs in hypercholesterolemia. Dietary flax seed supplementation could, therefore, prevent hypercholesterolemia-related heart attack and strokes.

ANTIOXIDANT DEFENSE SYSTEM IN DIABETIC KIDNEY : A TIME COURSE STUDY

Oxygen free radicals (OFRs) have been suggested to be a contributory factor in complications of diabetes mellitus. In the present study, we investigated the lipid peroxide level measured as thiobarbituric acid reactive substances (TBARS) and activities of antioxidant enzymes viz., [superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px)] in the kidney of streptozotocin induced diabetic rats at various stages of development of diabetes. Sprague Dawley rats were divided into two groups: group I, control (n = 42) and group II, diabetic (n = 42). Each group was further subdivided into seven groups each consisting of six rats. Rats in subgroups were studied at weekly intervals (0 to 6 weeks). Blood glucose levels were estimated at the time of sacrifice. TBARS levels and activity of antioxidant enzymes were measured in kidney. The levels of TBARS in the diabetic group increased initially, dropped to baseline level after 2 weeks and then progressively increased at 5th and 6th week (p < 0.05). There was an increase in catalase activity at first week after that it decreased as compared to control group. However, GSH-Px activity in the diabetic group increased after 1 week and then remained at the same level except a small drop in the 2nd week. Total SOD and CuZn-SOD activity increased significantly in diabetic kidney as compared to controls at all time intervals, while Mn-SOD activity showed no change. The present findings suggest that oxidative stress accompanies at early onset of diabetes mellitus and the susceptibility of the kidney to oxidative stress during the early stages may be an important factor in the development of diabetic nephropathy.

Reduction of hypercholesterolemic atherosclerosis by CDC-flaxseed with very low alpha-linolenic acid

Flaxseed (Type I flaxseed) with 51-55% alpha-linolenic acid in its oil and richest source of plant lignans, has been shown to reduce hypercholesterolemic atherosclerosis by 46% without lowering serum lipids. Antiatherogenic activity was claimed to be due to its alpha-linolenic acid and/or lignan content. If alpha-linolenic acid component of flaxseed is responsible for antiatherogenic activity, then, CDC-flaxseed (Type II flaxseed) which has similar oil and lignan content but has very little (2-3% of the total oil) alpha-linolenic acid would have no antiatherogenic effect. An investigation, therefore, was made of Type II flaxseed on high cholesterol diet-induced atherosclerosis and serum lipids [total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C)] in rabbits. Rabbits were assigned to four groups: Group I, Control; Group II, Type II flaxseed diet (7.5 g/kg orally daily); Group III, 1% cholesterol diet; Group IV, 1% cholesterol diet supplemented with Type II flaxseed (7.5 g/kg orally daily). Blood samples were collected before (0 time) and after 4 and 8 weeks of experimental diets for measurement of serum lipids. Aorta was removed at the end of 8 weeks for assessment of atherosclerotic plaques. Serum TC, LDL-C, TC/HDL-C, and LDL-C/HDL-C were lower in Group IV as compared to Group III by 14 and 31%, 17 and 32%, 28 and 34% and 24 and 32%, respectively, at 4 and 8 weeks. HDL-C was not affected by Type II flaxseed in hypercholesterolemic rabbit. TG and VLDL-C were markedly increased in Group IV as compared to Group III. Type II flaxseed reduced the development of atherosclerosis by 69%. Histological changes in the atherosclerotic regions were qualitatively similar in Groups III and IV. Results indicate that reduction in hypercholesterolemic atherosclerosis by Type II flaxseed is due to a decrease in serum TC and LDL-C. In conclusion, antiatherogenic activity of Type II flaxseed is not due to alpha-linolenic acid.

Antioxidant enzymes in hypercholesterolemia and effects of vitamin E in rabbits

We investigated the effects of high cholesterol diet in the absence and presence of vitamin E on the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px)] in rabbits. The animals were divided into 4 groups each comprising of 10 rabbits. Group I, regular rabbit chow diet; Group II, regular rabbit chow diet with added vitamin E; Group III, high cholesterol diet; and Group IV, high cholesterol diet+vitamin E. Antioxidant enzymes of blood were measured in each group before and after 1, 2, 3, and 4 months on the experimental diets. The aorta was removed at the end of the protocol for measurement of antioxidant enzymes. There was a decrease in activity of SOD and GSH-Px and an increase in activity of catalase in blood of Group III. Vitamin E produced a decrease in blood SOD, catalase and GSH-Px activity in Group II and prevented the decrease in SOD and GSH-Px activity in Group IV but did not affect the changes in the catalase activity. SOD, catalase and GSH-Px activity of aortae from Group III increased significantly, while catalase activity increased and GSH-Px activity decreased in those from Group II. Vitamin E prevented the cholesterol-induced rise in catalase and GSH-Px activity in aorta but did not prevent the rise in SOD activity. These results suggest that the activity of antioxidant enzymes in blood is affected differently from that in aortic tissue. There appears to be a mutually supportive interaction among the antioxidant enzymes which provide defense against oxidant injury. The protective effects of vitamin E against hypercholesterolemic atherosclerosis may not be due to changes in the antioxidant enzymes but may be mainly mediated through its chain-breaking antioxidant activity.