Effect of Semecarpus anacardium Linn. nut milk extract on rat neutrophil functions in adjuvant arthritis

Neutrophils play an important role in the pathogenesis of rheumatoid arthritis (RA) and various inflammatory conditions, by accumulation and liberation of active proteolytic enzymes. The effect of milk extract of Semecarpus anacardium Linn. nuts (SA) at a dosage of 150 mg kg(-1) body weight day(-1) for 14 days on adjuvant arthritis was studied to gain some insight into this intriguing disease in relation to neutrophil functions. The decreased phagocytic function of neutrophils (phagocytic index and avidity index) found in adjuvant arthritis was significantly increased by the administration of the drug SA. Increased levels of reactive oxygen species (superoxide radical, hydroxyl radical, H2O2 and myeloperoxidase), lysosomal enzymes (acid phosphatase and cathepsin D) and increased accumulation of neutrophils in the joints observed in adjuvant arthritic animals were reverted back to near normal levels by treatment with SA. The results of this study indicate that SA can be considered to be a good therapeutic agent for inflammation and arthritis.     

Salubrious effect of Kalpaamruthaa, a modified indigenous preparation in adjuvant-induced arthritis in rats--a biochemical approach

BACKGROUND: Interactions between the phytochemicals and drugs and their combinations are capable of providing longer remissions and perhaps a complete cure for many diseases including rheumatoid arthritis (RA). In addition to articular manifestations in RA, extra-articular signs involving reticuloendothelial and hepatic systems are an indication of more severe disease and thus, have prognostic value. OBJECTIVES: The present study was designed to illustrate the beneficial outcome of the drug Kalpaamruthaa (constituting Semecarpus anacardium nut milk extract, fresh dried powder of Emblica officinalis fruit and honey) in adjuvant-induced arthritic rat model with respect to the changes in extra-articular manifestation involving hematological and cellular constituents. MATERIAL AND METHODS: Levels of hematological parameters, cellular constituents, activities of marker enzymes and the level of DNA damage were assessed in control, arthritis-induced, SA, KA and drug control treated rats. RESULTS AND CONCLUSION: Significant decrease (p<0.005) in the levels of Hb, RBC, PCV, total protein, albumin, A/G ratio, plasma uric acid, urinary urea, uric acid, creatinine, FFA, HDL and significant increase (p<0.05) in the levels of WBC, platelet count, ESR, globulin, plasma creatinine, blood glucose, urea, AST, ALT, ALP, TC, FC, TG, PL, LDL and VLDL were observed in arthritic rats. No other significant change was observed in tissue DNA and RNA levels of control and experimental animals. On the contrary an increase in DNA damage was observed in arthritic rats when compared to control animals. The above said derangements were brought back to near normal levels upon SA and KA treatments and KA revealed a profound beneficial effect than SA. The enhanced effect of KA might be attributed to the combined effects of phytoconstituents such as flavonoids, tannins and other compounds such as vitamin C present in KA. Thus KA via this preliminary protective effect might contribute to the amelioration of the disease process.     

Curative effect of Semecarpus anacardium Linn. nut milk extract against adjuvant arthritis -- with special reference to bone metabolism

Localised bone loss in the form of bone erosions and peri-articular osteopenia constitutes an important criteria for the diagnosis of rheumatoid arthritis. In the present study, the effect of Semecarpus anacardium Linn. nut milk extract (SA) on the metabolism of bone turn over has been studied by analyzing various markers of bone turnover and by histological and radiological analysis of the joints in adjuvant arthritis in rats. Arthritis was induced in rats by injecting Freund's complete adjuvant containing 10mg of heat killed mycobacterium tuberculosis in 1 ml paraffin oil (0.1 ml) into the left hind paw of the rat intradermally. After 14 days of induction, SA (150 mg/kg body weight/day) was administered orally by gastric intubations for 14 days. SA significantly reverted the alterations in the bone turnover observed in arthritic animals by modulating the levels of calcium, phosphorus and the activities of the enzymes names tartrate resistant acid phosphatase, acid phosphatase and alkaline phosphatase. The drug increased the bone weights that were found to be decreased during arthritis. Protective effect of SA was also observed by the decrease in the levels and expression of tumour necrosis factor alpha (TNF-alpha) as well as the histopathological and radiological observations. From all these observations it can be concluded that SA possesses strong anti-arthritic property by regulating bone turnover.     

Apoptotic effect of Semecarpus anacardium nut extract on T47D breast cancer cell line

There is an increasing interest in identifying potent cancer-preventive and therapeutic agents against breast cancer. A great number of reports have in recent years dealt with anticancer characteristics of Semecarpus anacardium nut extract (SA). The majority of these studies has been targeted on the protective effect rendered to the living system rather than the preventive effect on cancer cells. SA was tested for its inhibitory effect on human breast cancer cells (T47D). Cytotoxicity analyses suggested that these cells had become apoptotic. SA was discovered to induce rapid Ca(2+) mobilization from intracellular stores of T47D cell line, and its cytotoxicity against T47D was well correlated with altered mitochondrial transmembrane potential. At the molecular level, these changes are accompanied by decrease in bcl(2) and increase in bax, cytochrome c, caspases and PARP cleavage, and ultimately by internucleosomal DNA fragmentation. Taken together, our results provide unprecedented evidence that SA triggers apoptotic signals in T47D cells.     

Therapeutic effects of Semecarpus anacardium Linn. nut milk extract on the changes associated with collagen and glycosaminoglycan metabolism in adjuvant arthritic Wistar rats

The effect of milk extract of Semecarpus anacardium Linn. nut milk extract (SA) was studied to gain some insight into this intriguing disease with reference to collagen metabolism. Arthritis was induced in rats by injecting Freund's complete adjuvant containing 10mg of heat killed mycobacterium tuberculosis in 1 ml paraffin oil (0.1 ml) into the left hind paw of the rat intradermally. After 14 days of induction, SA (150 mg/kg body weight/day) was administered orally by gastric intubations for 14 days. Decreased levels of collagen and glycosaminoglycans (GAGS) components (chondroitin sulphate, heparan sulphate, hyaluronic acid) and increase in the levels of connective tissue degrading lysosomal glycohydrolases such as acid phosphatase, beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin-D observed in arthritic animals were reverted back to near normal levels upon treatment with SA. The drug effectively regulated the uriniray markers of collagen metabolism namely hexosamine, hexuronic acid, hydroxyproline and total GAGS. Electron microscopic studies also revealed the protective effect of SA. Hence, it can be suggested that SA very effectively regulate the collagen metabolism that derange during arthritic condition.     

Age-related changes in antioxidant enzyme activities and lipid peroxidation in lungs of control and sulfur dioxide exposed rats

Antioxidant defenses within the lung are pivotal in preventing damage from oxidative toxicants. There have also been several reports with conflicting results on the antioxidant system during aging. In this study, we attempted to investigate age-related alterations in both antioxidant enzyme activities and thiobarbituric acid-reactive substances (TBARS), a product of lipid peroxidation, in the whole lung of control and sulfur dioxide (SO₂) exposed rats of different age groups (3-, 12-, and 24-months-old). Swiss-Albino Male rats were exposed to 10 ppm SO₂ 1 hr/day, 7 days/week for 6 weeks. The antioxidant enzymes examined include Cu,Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST). A mixed pattern of age-associated alterations in antioxidant activities was observed. SOD, GSH-Px and GST activities were increased with age, but CAT activity was decreased. Lung SOD, GSH-Px and GST activities were also increased in response to SO₂. The level of TBARS was increased with age. SO₂ exposure stimulated lipid peroxide formation in the lung as indicated by an increase in the level of TBARS. These findings suggest that both aging and SO₂ exposure may impose an oxidative stress to the body. We conclude that the increase in the activities of the antioxidant enzymes of the lung during aging, could be interpreted as a positive feedback mechanism in response to rising lipid peroxidation.     

催乳素对佐剂关节炎大鼠滑膜组织MMP-9表达的影响

目的：探讨催乳素（PRL）对佐剂关节炎（AA）大鼠滑膜组织基质金属蛋白酶-9（MMP-9）表达的影响,为进一步阐明PRL在类风湿性关节炎（RA）发病过程中的作用机制及为RA的治疗提供实验依据。方法：雄性Wistar大鼠32只分为4组（n=10）：①正常对照组（A组）（n=10）;②佐剂关节炎对照组（B组）（n=10）;③高催乳素血症佐剂关节炎组（C组）（n=10）;④低催乳素血症佐剂关节炎组（D组）。放射免疫法测定各组血清PRL含量;明胶酶谱法测定MMP-9的活性;免疫组织化学方法检测各组滑膜组织MMP-9免疫反应变化;Western blot测定各组滑膜组织MMP-9的表达。结果：B组滑膜组织MMP-9的活性及表达明显高于A组,C组MMP-9的活性及表达最高,D组较B组有所减少,但仍高于A组。结论：PRL影响滑膜组织MMP-9分泌是其影响RA发病过程的一种作用机制。     

Region specific increase in the antioxidant enzymes and lipid peroxidation products in the brain of rats exposed to lead

The objective of this study is to determine the effect of lead (pb) on antioxidant enzymes and lipid peroxidation products in different regions of rat brain. Wistar male rats were treated with lead acetate (500 ppm) through drinking water for a period of 8 weeks. Control animals were maintained on sodium acetate. Treated and control rats were sacrificed at intervals of 1st, 4th and 8th week and the whole brains were dissected on ice into four regions namely the cerebellum, the hippocampus, the frontal cortex and the brain stem. Antioxidant enzymes namely catalase and superoxide dismutase in all the four regions of brain were determined. In addition, lipid peroxidation products were also estimated. The results indicated a gradual increase in the activity of antioxidant enzymes in different regions of the brain and this response was time-dependent. However, the increase was more in the cerebellum and the hippocampus compared to other regions of the brain. The lipid peroxidation products also showed a similar trend suggesting increased effect of lead in these two regions of the brain. The data indicated a region-specific oxidative stress in the brain exposed to lead.     

蜜环菌提取物对佐剂性关节炎大鼠的治疗作用

本文通过对佐剂性关节炎大鼠原发性和继发性足肿胀度的测量、全身关节炎指数评分、体重及体态变化、免疫脏器指数、炎性踝关节切片的形态以及血清中肿瘤坏死因子α、白细胞介素1β、白细胞介素6、前列腺素E2、一氧化氮和一氧化氮合成酶等指标来考察蜜环菌Armillaria mellea的5个不同提取物对佐剂性关节炎大鼠的影响及作用机理。结果表明,蜜环菌甲醇提取物对原发性和继发性足肿胀有明显的抑制;甲醇组能明显抑制佐剂性关节炎大鼠脾脏和胸腺萎缩,并能抑制血清中肿瘤坏死因子α、白细胞介素1β、白细胞介素6和前列腺素E2的分泌,抑制大鼠踝关节炎细胞的形成。因此蜜环菌提取物中甲醇提取物对佐剂性关节炎大鼠治疗效果最显著。     

The biochemical alterations following administration of Kalpaamruthaa and Semecarpus anacardium in mammary carcinoma

BACKGROUND: Breast cancer is one of the most common cancers in women of developed and developing countries. Lipids, lipoproteins and lipid-metabolizing enzymes have been associated with the risk of breast cancer. Kalpaamruthaa (KA) is a modified Siddha preparation, which contains Semecarpus anacardium Linn. (SA), Emblica officinalis (EO) and honey. OBJECTIVE: The present study was embarked to study the variations in lipids, lipid-metabolizing enzymes and lipoproteins in cancerous animals and the effect of KA on the lipid metabolism. MATERIALS AND METHODS: Breast cancer was induced in rats by administrating 7,12-dimethylbenz(a)anthracene orally (25 mg/kg body weight). After 90 days of induction, KA (300 mg/kg body weight) and SA (200 mg/kg body weight) were administered for 14 days, by gastric intubations. The levels of lipids and lipid-metabolizing enzymes were analysed in control and experimental animals. RESULTS AND CONCLUSION: The increased levels of total cholesterol, free cholesterol, phospholipids, triglycerides and free fatty acids and decreased levels of ester cholesterol in plasma, liver and kidney found in cancer suffering animals were reverted back to near normal levels on treatment with KA and SA. In mammary carcinoma bearing animals, the activities of total lipase, cholesterol ester synthase, and cholesterol ester hydrolase were significantly (p < 0.05) increased whereas lipoprotein lipase and lecithin cholesterol-acyl transferase were decreased. The levels of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were increased and the level of high-density lipoprotein (HDL) was decreased. These alterations were recouped back upon treatment with KA as well as SA when compared to cancer animals. The effects of KA were found to be more effective than SA. No significant alterations were observed in herbal preparation control animals when compared to control animals.     

Plasma lipid peroxidation and antioxidant levels in patients with rheumatoid arthritis

In recent years, a great number of studies have investigated the possible role of reactive oxygen species in the aetiology and pathogenesis of rheumatoid arthritis (RA). The aim of this study was to investigate plasma concentrations of vitamin E, beta-carotene, activities of glutathione peroxidase (GSH-Px) and catalase, levels of lipid peroxidation (MDA) and reduced glutathione (GSH) in 36 patients with rheumatoid arthritis (RA) and 22 healthy age-matched controls. The plasma activity of GSH-Px and catalase (p < 0.001), levels of GSH (p < 0.01), concentration of beta-carotene (p < 0.05) and vitamin E (p < 0.001), haemoglobin and hematocrit (p < 0.05) were significantly lower in patients with RA than in controls. The MDA levels (p < 0.01), C reactive protein, rheumatoid factor, anti-streptolysin-o values (p < 0.001), platelet count (p < 0.05) and erythrocyte sedimentation rate (p < 0.001) were higher in the patient group than in the control group. These results provide some evidence for a potential role of increased lipid peroxidation and decreased enzymic and non-enzymic antioxidants in RA by its inflammatory character. These results suggested that oxidant stress plays a very important role in the pathogenesis of RA.     

Uniconazole-induced alleviation of freezing injury in relation to changes in hormonal balance, enzyme activities and lipid peroxidation in winter rape

Winter rape (Brassica napus L. cv. 601) seedlings were treated with 50 mg.l-1 of foliar-applied uniconazole and then exposed to freezing stress with a light/dark temperature regime of 2 °C/–3 °C for 5 days at the seedling stage. Stressed plants contained lower endogenous GA3 and IAA contents than the controls, while zeatin and ABA contents and ethylene levels were significantly increased. Uniconazole-treated plants had lower endogenous GA3 and IAA contents, and higher zeatin and ABA contents and ethylene levels. Leaf chlorophyll content and respiratory capacity of roots were reduced significantly after plants were subjected to freezing stress, and foliar sprays of uniconazole retarded the degradation of chlorophyll and increased respiratory capacity of roots. Uniconazole-induced freezing tolerance was accompanied by increased activities of various antioxidant enzymes, including superoxide dismutase, catalase and peroxidase. Foliar applications of uniconazole reduced electrolyte leakage and malondialdehyde accumulation caused by freezing stress, suggesting that uniconazole may have decreased freezing-induced lipid peroxidation and membrane damage.     

Plasma iron is associated with lipid peroxidation in an elderly population

Epidemiological evidence has raised concern that a moderate elevation in body iron stores may increase oxidative stress and risk of heart disease. We examined the cross-sectional association between plasma iron and factors that could affect its levels (antioxidant enzymes, diet), with the concentration of plasma malondialdehyde (MDA) as a marker of lipid peroxidation. Participants were 162 non-smoking institutionalised elderly. Our results show that those in the highest tertile of plasma iron were at least twice as likely to have higher plasma MDA levels. Among the factors affecting plasma iron levels, we found that the upper tertile of erythrocyte-superoxide dismutase (E-SOD) was inversely associated with higher plasma iron, and potato intake explained a sizeable proportion of the variation in plasma iron levels. In addition to potatoes, eggs, wine, fruit in men and green vegetables in women showed a positive association with plasma iron levels. Only potatoes in both sexes, wine in men and eggs in women had an independent effect on plasma MDA. Potatoes, wine, plasma lycopene and plasma iron accounted for 43% of the variability in plasma MDA for males, and E-SOD, potatoes, eggs, plasma lycopene and plasma iron explained 45% for women. A longitudinal study should confirm, whether these MDA levels are related to morbidity and mortality.     

Uniconazole-induced tolerance of rape plants to heat stress in relation to changes in hormonal levels, enzyme activities and lipid peroxidation

Winter rape (Brassica napus L. cv. 601) seedlings were treated with 50 mg.l-1of foliar-applied uniconazole and then exposed to heat stress with a light/dark temperature regime of 43 °C/38 °C for 3 days at the stem elongation stage. Heat stressed plants contained lower endogenous GA3, IAA and zeatin contents than the controls, while ABA content and ethylene level were increased significantly. Uniconazole-treated plants had lower endogenous GA3 and IAA contents, and higher zeatin and ABA contents and ethylene levels. Leaf chlorophyll content and respiratory capacity of roots were reduced markedly after plants were subjected to heat stress, and foliar sprays of uniconazole retarded the degradation of chlorophyll and increased respiratory capacity of roots. Following exposure to heat, the activities of superoxide dismutase and peroxidase were significantly reduced. Uniconazole-induced heat tolerance was accompanied by increased activities of various antioxidant enzymes. Foliar applications of uniconazole reduced electrolyte leakage and malondialdehyde accumulation caused by heat stress, suggesting that uniconazole may have decreased heat-induced lipid peroxidation and membrane damage. Foliar sprays of uniconazole increased the tolerance of rape plants to heat stress.     

Antioxidant status and lipid peroxidation in type II diabetes mellitus

Diabetes Mellitus (DM), a state of chronic hyperglycaemia, is a common disease affecting over 124 million individuals worldwide. In this study, erythrocyte glutathione levels, lipid peroxidation, superoxide dismutase, catalase, and glutathione peroxidase and some extracellular antioxidant protein levels of patients with type II diabetes mellitus and healthy controls were investigated. Thirty-eight patients (21 males; with age of mean +/- SD, 53.1+/-9.7 years) and 18 clinically healthy subjects (10 males; with age of mean +/- SD, 49.3+/-15.2 years) were included in the study. Levels of erythrocyte lipid peroxidation, serum ceruloplasmin and glucose levels, HbA1C levels, and erythrocyte catalase activity were significantly increased, whereas serum albumin and transferrin levels, erythrocyte glutathione levels, and glutathione peroxidase activity were significantly decreased compared to those of controls. There was no significant difference in superoxide dismutase activity compared to controls. The results suggest that the antioxidant deficiency and excessive peroxide-mediated damage may appear in non-insulin dependent diabetes mellitus.     

Hypoxia and its downstream targets in DMBA induced mammary carcinoma: protective role of Semecarpus anacardium nut extract

Tumors are usually exposed to a hypoxic microenvironment due to their irregular growth and abnormal vascular supply. Under hypoxia, gene regulation (selective activation and inactivation of genes) plays an important role in maintenance of tumor. Multiple hypoxic and angiogenic growth factors are expressed for tumor cell survival. In search of novel anticancer drug, Semecarpus anacardium nut extract (SA) was tried against breast cancer. Mammary carcinoma was induced in vivo by 7,12-dimethyl benz(a) anthracene (DMBA) (25mg/kg b.w., p.o.). Tumor development and vascular structures were accelerated by DMBA. Hypoxia inducible factor-1 alpha (HIF-1) was coexpressed with its downstream genes in mammary tissue. Cancer rats were then treated with S. anacardium nut extract (SA) (250mg/kg b.w., p.o.). Delay in the tumor growth was paralleled with a drastic reduction in vascularization by SA treatment. Activities of glycolytic enzymes were normalized with decreased expression of glucose transporter-1 and carbonic anhydrase IX by drug treatment. Inhibition of HIF-1, vascular endothelial growth factor and inducible nitric oxide synthase by SA may in part explain its antiangiogenic action. SA also inhibits endothelial cell proliferation by blocking the overexpressed survival cytokines. In conclusion, our study demonstrates that at least some part of the antitumor activity of SA is due to the suppression of hypoxic and angiogenic factors. The mechanism of this inhibition seems to be through an action of SA on expression of HIF-1 and its downstream targets.     

Role of zinc on lipid peroxidation and antioxidative enzymes in intestines of ethanol-fed rats

This study was undertaken to investigate the effects of zinc on lipid peroxidation and various antioxidative enzymes in the intestines of male Wistar rats fed on ethanol. It was observed that NADPH-dependent lipid peroxidation (LP) was significantly increased upon ethanol treatment for 4 and 8 wk. The concentraton of glutathione as well as the activities of catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were also found to be significantly increased upon ethanol feeding at all of the treatment intervals. The glutathione levels were found to be further elevated upon combined zinc and ethanol treatments. Interestingly, the administration of zinc to ethanol-fed rats was able to bring down the elevated levels of LP, catalase, SOD, and GPx, thus indicating the antiperoxidative potential of zinc under such conditions.     

Effect of lead exposure on liver lipid peroxidative and antioxidant defense systems of protein-undernourished rats

The effect of the liver mitogen, lead nitrate [Pb(NO₃)₂], on protein-undernutrition-induced increased lipid peroxidation and reduced antioxidants levels was investigated in rats. Animals were divided into four groups: A, B, C, and D of five animals each. Animals in groups C and D were placed on a low-protein diet (5% casein) and animals in groups A and B were maintained on a normal diet (16% casein) for 14 wk and fed ad libitum. Animals in groups B and D were each given a single intravenous injection of Pb(NO₃)₂ (100 μmol/kg body weight) 72 h before sacrifice. The results confirm that protein undernutrition (PU) induced an increase in lipid peroxidation with concomitant reductions in catalase (CAT) activity, glutathione (GSH) level, and superoxide dismutase (SOD) activity. Lead (Pb) treatment, however, provoked increased lipid peroxidation, CAT activity, and GSH level but resulted in reduced SOD activity in both normal and PU-rats. These results suggest that Pb exacerbates liver lipid peroxidation in PU rats and suggests the involvement of free radicals in the pathogenesis of Pb poisoning. In addition, the results show that Pb affects well-fed and PU rats in similar ways but that the CAT activity of PU rats is more sensitive to the effect of Pb than that of normal rats.     

Interaction of Semecarpus anacardium L. with propranolol against isoproterenol induced myocardial damage in rats

With a view to evaluate the cardioprotective effect of ethanolic extract of S. anacardium nut and the possible interaction with propranolol against isoproterenol induced myocardial damage in rats, female Sprague-Dawley rats were pre-treated with propranolol (10 mg/kg for 7 days), low and high doses of S. anacardium (100 and 500 mg/kg for 21 days) and their combination orally and subsequently subjected to isoproterenol administration (150 mg/kg, sc) for two consecutive days. The influence of prophylactic treatment was analysed by quantification of biomarkers and antioxidants, electocardiographic parameters and histopathological observations. The activities of lactate dehydrogenase and creatinine phosphokinase-MB were reduced in serum and raised in heart tissue with concurrent elevation in superoxide dismutase and catalase activities as well as reduction in thiobarbituric acid reactive species levels significantly in all treated groups compared to isoproterenol group. Similarly, electrocardiographic changes were restored to normalcy in all treated groups. To conclude, combination of high dose of S. anacardium with propranolol was found to be most effective in alleviating the abnormal conditions induced by isoproterenol.     

Effect of isoproterenol on lipid peroxidation and antioxidant enzymes of myocardial tissue of mice and protection by quinidine

administration of isoproterenol to mice at a dose of 30 mg/100 g body weight for 3 consecutive days at an interval of 24 h induced lipid peroxidation in cardiac tissue and exhibited a significantly elevated serum glutamate oxaloacetate transaminase (SGOT) level. Increased superoxide dismutase (SOD) activity with a concomitant decrease in catalase activity has also been observed in cardiac tissue with isoproterenol treatment. Quinidine, a class I antiarrhythmic agent has been found to exhibit a protective role in isoproterenol induced myocardial ischaemia. Cardiac tissue of quinidine treated mice showed reduction of lipid peroxidation reaction. In addition, quinidine treatment is found to influence the cardiac antioxidant enzymes – catalase and SOD. The decrease of SOD activity and increase of catalase activity suggests that quinidine also exerts an indirect antioxidant effect in protecting the myocardial tissue from reactive oxygen species. Furthermore, our current in vitro studies with quinidine have clearly shown in this work that it possesses a very convincing hydroxyl radical scavenging potential with almost no ability to scavenge superoxide anion and hydrogen peroxide (H₂O₂) in vitro. Thus, our present investigation suggests that quinidine, when administered to mice, strengthens the antioxidant defense system to resist the free radical induced damage brought about by isoproterenol induced ischaemic condition.