Oxidative Stress and C-Reactive Protein in Patients with Cerebrovascular Accident ( Ischaemic Stroke ) The role of Ginkgo biloba extract *

Objectives: This study aimed to investigate the presence of oxidative stress and inflammation in ischaemic stroke patients by measuring malondialdehyde (MDA), total antioxidant status (TAS), and highlysensitivity C-reactive protein (hsCRP) in the early post-ischaemic period, and to determine the role of Ginkgo biloba therapy in correcting the markers of oxidative stress and inflammation. Methods: This study was conducted at Ibn Seena Hospital, Mosul City, Iraq and included 31 cerebrovascular accident (CVA) patients and 30 healthy controls. Ischaemic stroke patients were divided into two groups: group I (n = 15) received conventional therapy; group II (n = 16) received conventional therapy with G. biloba (1500 mg/day) for 30 days. Blood samples were obtained from patients and controls before treatment and assays done of serum levels of MDA, TAS, and hsCRP. For CVA patients, a post-treatment blood sample was taken and the same parameters reassessed. Results: Compared with the controls, patients’ serum levels of MDA, and hsCRP were significantly higher (P ≤0.001) and TAS significantly lower. Group I and II patients reported a significant reduction in serum levels of MDA and hsCRP and a significant increase in serum levels of TAS, in comparison with pre-treatment levels. There was no significant difference (P = 0.19) in serum MDA levels between groups I and II, whereas, serum TAS levels were significantly higher (P ≤0.01) and hsCRP significantly lower (P ≤0.01) in group II. Conclusion: Acute stroke is associated with oxidative stress and inflammatory response in the early period. G. biloba plays a potential role in reducing oxidative damage and Oxidative Stress and C-Reactive Protein in Patients with Cerebrovascular Accident (Ischaemic Stroke) The role of Ginkgo biloba extract *Imad A-J Thanoon,1 Hilmy AS Abdul-Jabbar,2 Dhia A Taha3 CLINICAL & BASIC RESEARCH Oxidative Stress and C-Reactive Protein in Patients with Cerebrovascular Accident (Ischaemic Stroke) The role of Ginkgo biloba extract 198 | SQU Medical Journal, May 2012, Volume 12, Issue 2 Stroke, or cerebrovascular accident (CVA), is the third leading cause of death after cardiovascular diseases and cancer.1 In fact, it is the second-leading cause of mortality and disease among adults over 60 years of age worldwide.2 Approximately 80% of strokes are ischaemic in origin, since they result either from thrombus in situ or an embolism of distant origin.3 Cerebral ischaemia initiates a cascade of cellular and molecular events that lead to brain damage, with one such event being post-ischaemic inflammation.4 Focal cerebral ischaemia is associated with a local inflammatory reaction that contributes to tissue damage.5 Microglial cells in particular become activated and provoke tissue injury by releasing pro-inflammatory mediators and reactive oxygen species (ROS).6,7 When oxygen supply is limited during ischaemia, a calcium influx may activate phospholipase C, which results in a breakdown of membrane phospholipids, or may convert xanthine dehydrogenase to xanthine oxidase in the cerebral blood vessels leading to the formation of superoxide radicals and hydrogen peroxide.8 ROS causes oxidative damage that may affect lipids, proteins, nucleic acids and other molecules. Quantification of lipid peroxidation end products is considered to be a measure of whole-body oxidative damage. Serum malondialdehyde (MDA), a marker of lipid peroxidation, is the most abundant aldehyde generated by the attack of free radicals on polyunsaturated fatty acids of cell membranes; its measurement provides information of oxidative injury in vivo.9 The impact of free radicals may also be obtained by comparisons of antioxidant concentrations, because serious damage by free radicals implies insufficiency of the body’s multilevel defence systems against radicals.10Measurement of the total antioxidant capacity (TAC) of biological fluids, however, is regarded as more physiologically representative in certain settings than individual antioxidants, and is believed to be a useful measure of how much the antioxidants present can protect against oxidative damage to membranes and other cellular components.11 C-reactive protein (CRP) has been the most extensively studied marker of inflammation. It is a novel plasma marker of atherothrombotic disease.12 C-reactive protein (CRP) is produced not only by the liver but also in atherosclerotic lesions by vascular smooth muscle cells and macrophages in response to stimulation by the ‘pro-inflammatory’ cytokine interleukin-6 (IL-6).13,14 Elevated plasma levels of CRP are not disease specific but are sensitive markers which are produced in response to tissue injury, infectious agents, and inflammation.12 Various cross-sectional studies support the notion that CRP may be a marker for stroke and poststroke status.15,16 Several studies support the role of CRP in the prediction of ischaemic stroke risk and outcome, as well as the possible role of inflammation before and after stroke.17,18As the methods traditionally employed to measure CRP do not have good sensitivity, measurement of highly-sensitivity C-reactive protein (hsCRP) is recommended to evaluate atherothrombotic disease, which usually presents with lower CRP levels than the other inflammatory processes.14 Ginkgo biloba extract (EGb 761) is known to have neuroprotective properties in diseases associated with free radical generation. Extensive studies on G. biloba extracts showed their ability to protect brain neurons from oxidative stress19 and to inhibit apoptosis in cell culture.20 The G. biloba extracts that are currently used for medicinal purposes contain 24% flavonoid glycosides (quercetin, kaempferol, isorhamnetin) and 6% terpene lactones (ginkgolides A, B, C, M, J and bilobalides).21 The EGb 761 components eliminate free radicals Advances in Knowledge Ischaemic stroke is associated with oxidative stress and inflammatory responses in the early post-ischaemic period. Applications to Patient Care Ginkgo biloba therapy plays a potential role in reducing oxidative damage and inflammatory response in early post-ischaemic stroke patients. inflammatory response.


S
troke, or cerebrovascular accident (CVA), is the third leading cause of death after cardiovascular diseases and cancer. 1 In fact, it is the second-leading cause of mortality and disease among adults over 60 years of age worldwide. 2Approximately 80% of strokes are ischaemic in origin, since they result either from thrombus in situ or an embolism of distant origin. 3erebral ischaemia initiates a cascade of cellular and molecular events that lead to brain damage, with one such event being post-ischaemic inflammation. 4ocal cerebral ischaemia is associated with a local inflammatory reaction that contributes to tissue damage. 5Microglial cells in particular become activated and provoke tissue injury by releasing pro-inflammatory mediators and reactive oxygen species (ROS). 6,7When oxygen supply is limited during ischaemia, a calcium influx may activate phospholipase C, which results in a breakdown of membrane phospholipids, or may convert xanthine dehydrogenase to xanthine oxidase in the cerebral blood vessels leading to the formation of superoxide radicals and hydrogen peroxide. 8OS causes oxidative damage that may affect lipids, proteins, nucleic acids and other molecules.Quantification of lipid peroxidation end products is considered to be a measure of whole-body oxidative damage.Serum malondialdehyde (MDA), a marker of lipid peroxidation, is the most abundant aldehyde generated by the attack of free radicals on polyunsaturated fatty acids of cell membranes; its measurement provides information of oxidative injury in vivo. 9The impact of free radicals may also be obtained by comparisons of antioxidant concentrations, because serious damage by free radicals implies insufficiency of the body's multilevel defence systems against radicals. 10Measurement of the total antioxidant capacity (TAC) of biological fluids, however, is regarded as more physiologically representative in certain settings than individual antioxidants, and is believed to be a useful measure of how much the antioxidants present can protect against oxidative damage to membranes and other cellular components. 11-reactive protein (CRP) has been the most extensively studied marker of inflammation.It is a novel plasma marker of atherothrombotic disease.12 C-reactive protein (CRP) is produced not only by the liver but also in atherosclerotic lesions by vascular smooth muscle cells and macrophages in response to stimulation by the 'pro-inflammatory' cytokine interleukin-6 (IL-6).13,14 Elevated plasma levels of CRP are not disease specific but are sensitive markers which are produced in response to tissue injury, infectious agents, and inflammation.12 Various cross-sectional studies support the notion that CRP may be a marker for stroke and poststroke status.8 As the methods traditionally employed to measure CRP do not have good sensitivity, measurement of highly-sensitivity C-reactive protein (hsCRP) is recommended to evaluate atherothrombotic disease, which usually presents with lower CRP levels than the other inflammatory processes.14 Ginkgo biloba extract (EGb 761) is known to have neuroprotective properties in diseases associated with free radical generation.Extensive studies on G. biloba extracts showed their ability to protect brain neurons from oxidative stress 19 and to inhibit apoptosis in cell culture.20 The G. biloba extracts that are currently used for medicinal purposes contain 24% flavonoid glycosides (quercetin, kaempferol, isorhamnetin) and 6% terpene lactones (ginkgolides A, B, C, M, J and bilobalides).21 The EGb 761 components eliminate free radicals such as the hydroxyl radical and the superoxide anion.22 Quercetin is a powerful antioxidant in the flavonoid family due to its molecular configuration, which is capable of eliminating free radicals. 23 Th pharmacologically active terpene lactones selectively inhibit the platelet-activating factor, preventing thrombus formation.Bilobalide is reported to possess neuroprotective properties.24 The aim of the present study was to investigate the presence of oxidative stress and inflammation in serum samples of ischaemic stroke patients by measuring MDA concentrations, total antioxidant status (TAS), and hsCRP in the early post-ischaemic period, and to determine the role of G. biloba therapy in correcting the markers of oxidative stress and inflammation in question.All patients included in this study were initially diagnosed as having CVA, or acute ischaemic stroke.All had problems with anterior circulation, a diagnosis made on the basis of full physical and neurological examinations by a neurologist. Thdiagnoses were then confirmed by either a magnetic resonance imaging (MRI) or computed tomography (CT) scan of the brain.Vascular risk factors including hypertension or diabetes mellitus, and smoking and alcohol habits were recorded.Patients with haemorrhagic stroke, intracranial tumour, or other neurological diseases, infection, inflammation, liver disease, and renal failure were excluded.For controls, the criteria were as follows: age ≥60 years, healthy subjects, non-smokers, and not taking vitamin supplements.

Methods
Ischaemic stroke patients were divided into two groups: group I (n = 15 of a possible 18 patients) included patients with ischaemic stroke who received conventional therapy (aspirin, rosuvastatin and lisinopril), and group II (n = 16 of a possible 28 patients) included patients who received conventional therapy with G. biloba (1500 mg/ day) for 30 days.The dose was decided as a safe increment after previous promising results with 500 and 1000 mg/day.
Blood samples were initially obtained from all CVA patients within 48-120 hours of their accidents, and before starting treatment.They were also taken from the controls.Assays of the serums MDA, TAS and hsCRP were done at the Department of Pharmacology in the College of Medicine at the University of Mosul.For the patient group, another blood sample was taken after treatment with either conventional therapy or conventional therapy with G. biloba, and the parameters reassessed.
MDA was measured by the method outlined by Buege and Aust where MDA reacts with thiobarbituric acid (TBA) to yield a red-coloured product. 25The absorbance of a 3 ml coloured layer was measured at 535 nm spectrophotometrically.TAS was measured by peroxidase/H 2 O 2 /ABTS colorimetric assay using commercial kits from Randox Laboratories, Belfast, UK.CRP was measured using the BioCheck hsCRP ELISA Kit (BioCheck, Inc., Foster City, California, USA).
Data were expressed as means ± standard deviation (SD).Statistical comparisons were performed using the Student's t-test between patients before therapy and controls, and the oneway analysis of variance (ANOVA).The Dunnett test was used to compare groups of patients.Linear regression analysis and Pearson correlation coefficients (r) were performed to determine the relationships between parameters.All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) for Windows (Version 11.5, Chicago, Illinois, USA).A P value of

Results
The serum levels of MDA, TAS and hsCRP from healthy subjects and patients with acute ischaemic stroke before starting drug therapy are shown in Table 1.The serum levels of MDA and hsCRP were found to be significantly higher (P ≤0.001) and TAS were significantly lower in ischaemic stroke patients in the early post-ischaemic period (before starting therapy) in comparison to the controls [Table 1].
Table 2 shows the serum levels of MDA, TAS, and hsCRP in the two groups of ischaemic stroke patients before and after therapy.Patients in group I reported a significant reduction in serum levels of MDA (P ≤0.001) and hsCRP (P ≤0.01), and a significant increase (P ≤0.01) in serum levels of TAS after treatment with conventional therapy in comparison with their levels before therapy.Also, patients in group II reported a significant reduction in serum levels of MDA (P ≤0.001) and hsCRP (P ≤0.001) and a significant increase in serum levels of TAS (P ≤0.001) after treatment with G. biloba together with conventional therapy in comparison with their levels before therapy.
By comparing the serum levels of MDA, TAS, and hsCRP between the two groups of ischaemic stroke patients after therapy, no significant differences (P = 0.19) were reported in serum MDA levels between group I, who received conventional therapy, and group II, who received G. biloba with conventional therapy, whereas the serum levels of TAS were found to be significantly higher (P ≤0.01) and hsCRP were significantly lower (P ≤0.01) in group II who received G. biloba with conventional therapy in comparison with group I who received conventional therapy alone [Table 2].
Regarding correlations between different biochemical parameters, Figures 1 to 3 show the relationships between MDA, TAS, and hsCRP in patients with acute ischaemic stroke.A significant negative correlation (r = -0.418,P = 0.001) was observed between MDA and TAS in the serum samples of patients with acute ischaemic stroke.MDA had a significant positive correlation with hsCRP (r = 0.729, P ≤0.001), and there was a significant negative correlation (r = -0.602,P = <0.001) between TAS and hsCRP in the serum samples of ischaemic stroke patients.

Discussion
There is strong indirect evidence that free radical production appears to be an important mechanism of brain injury after exposure to ischaemia and reperfusion. 26Free radicals in biological samples are difficult to measure because they are extremely reactive and have a short half-life.Therefore, particularly in human studies, indirect approaches have been used to demonstrate free radical production during cerebral ischaemia by measuring the products of free radical reaction with other molecules, such as lipids, proteins, and deoxyribonucleic acid (DNA), and the level or activity of antioxidant molecules. 27,28ROS causes impairment of cellular membrane stability and cell death by lipid peroxidation. 29MDA is the end product of the lipid peroxidation process. 30An increase in free radicals causes overproduction of MDA, which is commonly known as a marker of oxidative stress. 31In the present study, it was observed that ischaemic stroke patients in the early postischaemic period (before starting therapy) had significantly higher levels of serum MDA and hsCRP, and significantly lower TAS than controls.These findings provide evidence for the presence of oxidative stress and inflammation in ischaemic stroke patients.
There are several possible reasons for increased lipid peroxidation in cases of ischaemic stroke.First, the brain's cellular membranes are very rich in polyunsaturated fatty acid side chains, which are especially sensitive to free radical attack.Additionally, they have a low content of antioxidant enzymes, such as catalase and glutathione peroxidase, while the brain contains a significant amount of iron, despite the fact that its iron binding capacity is not very high.Iron ions are known to stimulate free radical generation. 32,33Lower TAS was accounted for by an increased use of endogenous antioxidants to fight free radicals and oxidative stress during ischaemic stroke. 34ur findings are in accordance with several studies which have been done to evaluate oxidative stress, antioxidant status, and markers of inflammation in ischaemic stroke patients. 5-8Most of these studies have shown enhanced levels of oxidative stress which are markers of inflammation, and reduced levels of antioxidants; 5,10 however, some studies have reported controversial and conflicting results with regard to the levels of antioxidant enzymes. 35][38][39] Some studies showed persistently elevated MDA concentrations 6 months after strokes. 40valuations of antioxidants in blood, urine, or cerebrospinal fluid of ischaemic stroke patients revealed lower plasma vitamin C, E, vitamin A, and uric acid, lower glutathione peroxidase (GPX) activity, decreased glutathione (GSH) concentration, and a decreased total plasma antioxidant capacity. 8,10,32,34,35,37,41ata associated with superoxide dismutase (SOD) activity after acute ischaemic stroke are controversial.SOD activity of patients with acute ischaemic stroke was reported to be reduced in the serum and increased in the cerebrospinal fluid (CSF), increased in both CSF and serum, or remained unchanged. 32,24,43everal prospective studies have shown that an elevated serum CRP concentration is a strong predictor of cardiovascular events, including stroke.CRP plays an important role as a marker of outcome and may determine the degree of recovery for stroke patients.
In their study, Sánchez-Moreno et al. found that ischaemic stroke patients had significantly  The relationship between malondialdehyde (MDA) and total antioxidant status (TAS) in patients with acute ischaemic stroke (r = -0.418,P = 0.001) elevated markers of inflammation, marked by CRP, intracellular adhesion molecule-1 (ICAM-1), and chemokine monocyte chemottractant protein-1 (MCP-1), and that elevated CRP concentrations were associated with a two-fold increase in the risk of ischaemic stroke.Winbeck et al. observed that an increase in CRP levels between 12 and 24 hours after the onset of symptoms predicts an unfavorable outcome and is associated with an increase in the incidence of cerebrovascular events.Mishra et al. observed an increase in hsCRP levels in stroke patients and that the increased levels were correlated with larger infarct, severe neurological deficit, and worse outcomes. 12ur study showed a highly significant negative correlation between serum MDA levels and TAS in ischaemic stroke patients, which suggests increased utilisation by ROS as an important contributing factor to the lower concentrations of antioxidants in ischaemic stroke patients.
In the present study, we have investigated the relationship between markers of oxidative stress and markers of inflammation in ischaemic stroke patients.Ischaemic stroke patients with higher serum hsCRP concentrations, indicative of greater inflammatory response, also had higher serum MDA levels and lower TAS than those with lower serum hsCRP concentrations.This can be observed in the figures, which show that serum hsCRP levels positively correlate with lipid peroxidation products and negatively correlate with TAS.
Our findings are in accordance with Sánchez-Moreno et al.'s results in which they found that CRP were inversely associated with concentrations of antioxidant vitamins C and E, and positively associated with markers of oxidative stress (8-isoprostanes). 41his study also showed that patients in groups I and II reported a significant reduction in serum levels of MDA and hsCRP and a significant increase in serum levels of TAS after treatment in comparison with their levels before therapy.
Interestingly, we did not observe any statistically significant differences in serum MDA levels between group I, who received conventional therapy, and group II, who received G. biloba with conventional therapy.In fact, the serum levels of TAS were found to be significantly higher, while those of hsCRP were significantly lower in group II who received G. biloba with conventional therapy, in comparison with group I who received conventional therapy alone.
Higher TAS in the serum of ischaemic stroke patients who received G. biloba together with conventional therapy (group II) was most likely caused by a lower use of endogenous antioxidants because of supplementary antioxidant effects of flavonoids glycosides. 23.
Antioxidant treatment may be an efficient therapeutic option for cardiac embolisms and macroangiopathic strokes, contributing to an improvement of neurological deficits and the functional status of the patients through the reduction of oxidative stress following ischaemia  and/or reperfusion. 48t has been reported in many studies that G. biloba improved tissue damage in various organs through its antioxidant effect.Zeybek et al. reported that G. biloba significantly decreased MDA levels and histopathological scores of the pancreatitis in rats. 49Bridi et al. reported that G. biloba had antioxidant activity in the hippocampus, striatum, and substantia nigra of rats. 50EGb 761 has an antioxidant effect as a free radical scavenger, a relaxing effect on vascular walls, an ameliorating effect on blood flow and microcirculation, and a stimulating effect on neurotransmitters.Besides a direct scavenging effect on ROS, G. biloba exerts an anti-inflammatory effect on inflammatory cells by suppressing the production of ROS and nitrogen species. 51everal studies have shown that EGb 761 could protect cultured neurons against damage induced by peroxynitrate and hydrogen peroxide. 19,51Zhang et al. demonstrated that total ginkgolides (TG) (a terpenoid constituent of EGb 761) protected cultured rat cortical neurons from oxidative damages induced by hydrogen peroxide (H 2 O 2 ). 52Gb 761 extract, with free radical scavenging activity, has been shown to reduce the size of cerebral infarction and improve neurological behaviour in rats with permanent and transient mid-cerebral artery occlusion (MCAo). 53,54aleem et al. showed that the standardised EGb761 significantly improved the outcome in mice after cerebral ischaemia and reperfusion in terms of neurobehavioral function and infarct size without affecting physiological parameters. 55ur findings suggest a potential role of G. biloba in acute ischaemic stroke, and the findings are important in view of the fact that stroke is, at present, the third leading cause of death worldwide. 1he mechanisms by which G. biloba normalise the cerebral damage, and reduce oxidative stress and inflammation, can probably be attributed to the antioxidant effects of flavonoids combined with the anti-inflammatory properties of the terpenoids bilobalide and ginkgolides A, B, C, M, J, and the terpenoids antagonists of platelet-activating factor (PAF), making this natural extract plausible to use in the treatment of ischaemic stroke, which is characterised by both oxidative damage and inflammation. 56ne of the limitations of this study is that it does not relate the biochemical changes to the clinical evaluation and outcome prognosis.

Conclusion
From this study, we conclude that acute ischaemic stroke is associated with oxidative stress and inflammatory response as indicated by increased lipid peroxidation products (MDA), reduced TAS and elevated levels of hsCRP in the early postischaemic period, and that G. biloba therapy has a potential role in reducing oxidative damage and inflammatory response in ischaemic stroke patients.

Table 1 :
Concentrations of malondialdehyde (MDA), total antioxidant status (TAS), and highly-sensitivity C-reactive protein (hsCRP) in patients with acute ischaemic stroke before therapy and in healthy controls

Table 2 :
Concentrations of malondialdehyde (MDA), total antioxidant status (TAS), and highly-sensitivity C-reactive protein (hsCRP) in patients with acute ischaemic stroke before therapy and after therapy