Int J Pharm Pharm Sci, Vol 8, Issue 8, 237-242Original Article


NEURO-PROTECTIVE EFFECTS OF GINKGO BILOBA LEAVES EXTRACT ON CEREBRAL ISCHEMIA-REPERFUSION INJURY INDUCED EXPERIMENTALLY IN OVARIECTOMIZED RATS

RASHA E. MOSTAFA*, BASSANT M. M. IBRAHIM, GEHAD A. ABDEL JALEEL

Pharmacology Department, Medical Division, National Research Centre, Cairo, Egypt
Email: dr_rosha81@yahoo.com

Received: 25 Apr 2016 Revised and Accepted: 20 June 2016

ABSTRACT

Objective: This study investigated the possible neuroprotective effects of Ginkgo biloba on cerebral ischemia-reperfusion (I/R) injury in ovariectomized rats.

Methods: Rats were randomly allocated into 5 groups as follows: Sham-operated group, Ovariectomized group (OVX), Cerebral I/R injury ovariectomized group (OVX+I/R) and two treated groups given Ginkgo biloba leaves extract in two doses [(OVX+I/R)+Gin50 and (OVX+I/R)+Gin100] for one month starting one month after ovariectomization. Two months after ovariectomization, cerebral I/R injury was induced. 24 h later, blood and brain samples were collected.

Results: Ginkgo biloba (50 and100 mg/kg) significantly decreased concentrations of TNF-α, MDA and NO, increased GSH concentration in serum and brain tissue and improved the histopathological pictures of treated rats when compared to ovariectomized cerebral I/R injury group. The effect of Ginkgo biloba high dose was better than the low dose.

Conclusion: Thus the study suggests Ginkgo biloba as a potent supplement for protection against cerebral I/R injury in estrogen deficient females, after full clinical evaluation. The proposed mechanisms include neuroprotective, anti-inflammatory and anti-oxidant actions.

Keywords: Ginkgo biloba, Cerebral, Ischemia-Reperfusion injury, Ovariectomy, Rats

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Premenopausal females exhibit a lower susceptibility to many cardiovascular and cerebrovascular diseases than males and postmenopausal females. This apparent protection is diminished and even lost, within ten years of menopause [1]. Brain damage after stroke in female animals is less when compared with their age-matched males, and this beneficial effect disappears with depletion of endogenous ovarian steroidal hormones [2]. Neuroprotection in premenopausal females may be attributed to higher levels of circulating estrogens; principally 17b-estradiol (E2). E2 is the most potent endogenously synthesized and secreted ovarian estrogen. During aging; neuroinflammation is a common feature of many cerebrovascular disorders, and is often associated with the release of pro-inflammatory cytokines and chemokines [3]. This neuroinflammatory response is referred to as “inflamm-aging” [4]; where blood levels of IL-6 and TNF-α significantly increase [5, 6], causing chronic subclinical inflammation that may lead to neurodegeneration and cognitive decline [7]. In young and middle-aged ovariectomized rodents, a model used to mimic surgical menopause, pro-inflammatory cytokine production increased in several injury models in both the central and peripheral nervous systems, whereas treatment with E2 attenuated this increase in cytokine production [8, 9]. Furthermore, inflammatory changes produced during menopause can stimulate innate immune responses in the brain and aggravate ischemic damage [10]. Despite the fact that the risk of cerebrovascular events rises in women after menopause [11], and that the efficacy of postmenopausal estrogen replacement therapy (ERT) in stroke prevention is still controversial, However, ERT successfully reduces the risk of cardiovascular disease [12]. The anti-inflammatory activities of E2 are mainly mediated in the brain tissue by the estrogen receptors (ER); ERα and ERβ [13, 14].

Mechanical recanalization or thrombolysis leads to restoration of blood flow following ischemic stroke [15]. Unfortunately, reperfusion may exacerbate the ischemic injury in some patients, causing what is known as “Cerebral Reperfusion Injury”. Cerebral reperfusion injury is the deterioration of ischemic but salvageable brain tissue after reperfusion [16, 17]. The pathogenesis of this injury includes post-ischemic hyper-perfusion, destruction of the blood–brain barrier, leukocyte infiltration and\or platelet activation [18].

Ginkgo (the maidenhair tree) is among the oldest living species of trees on earth and that's why it is called the "living fossil". Ginkgo belongs to the family Ginkgoaceae that flourished in large forests over 150 million years and is currently grown around the world for purposes [19]. Ginkgo tree leaves and seeds have been used for medicinal purposes since 1509 A. C. In traditional Chinese medicine, the extract of Ginkgo leaves has been used to treat circulatory disorders, cognitive problems, asthma, tinnitus and vertigo [20]. Highly-concentrated and stable extracts from Ginkgo leaves, have been extracted and standardized successfully, in the early 1970s by Dr. Willmar Schwabe Pharmaceuticals (Karlsruhe, Germany) [21]. The constituents of the standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, are as follows: 24% flavonoid glycosides, 6% terpenoids and 5–10% organic acids. The pharmacologically active constituents of EGb761 are flavonoids and terpenoids [22, 23]. Nowadays, film-coated tablets, oral liquids or injectable solutions of EGb761 can be purchased and are one of the most commonly taken herbal medicines worldwide [24]. In Germany EGb761 has been approved for treatment and prevention of cerebral insufficiency, neurodegenerative dementias associated with ageing as Alzheimer’s disease, peripheral vascular diseases, intermittent claudication, neurosensory problems as tinnitus and vertigo [25, 26]. Ginkgo is also called the "Brain Herb," it has been studied for the treatment of cerebral atherosclerosis, cerebral insufficiencies and depression [27]. The role of EGb761 in neuropsychopharmacology has been demonstrated by Dua et al. (2009)[28]. Many studies revealed that EGb761 has antioxidant and free radical scavenging effects [29]. EGb761 directly attenuates reactive oxygen species (ROS) and stabilizes the cellular redox state by up-regulating the activity of antioxidant enzymes [30]. Moreover, EGb761 enhances the activity of glutathione reductase and gamma-glutamylcysteinyl synthetase, the main enzymes essential for reduction and synthesis of glutathione (GSH) [31]. Also anti-apoptotic, anti-inflammatory activity [32, 33], protection against mitochondrial dysfunction [34, 35], and induction of growth factors [36] are proposed mechanisms of action of EGb761.

Therefore, the aim of the current study is to elucidate the neuroprotective effects of Ginkgo biloba leaves extract (EGb761) on cerebral ischemia-reperfusion injury induced experimentally in ovariectomized rats.

MATERIALS AND METHODS

Animals

Adult female albino rats, weighing 250-280 g, were used in the present study. The animals were obtained from the Animal House Colony of National Research Center (Dokki, Cairo, Egypt) and were housed under conventional laboratory conditions throughout the period of the experimentation. Animals were provided with standard laboratory food pellets and tap water ad libitum. The study was conducted in accordance with the National Research Centre-Medical Research Ethics Committee (NRC-MREC) for the use of animal subjects and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals [37].

Rats were anesthetized with thiopental sodium (20 mg/kg; ip) and then ovariectomized according to the method described by Turner et al. (2000) [38]. Sham-operated control group of animals was also included. Drug administration was started one month after ovariectomization and continued for another month.

Drugs

Ginkgo biloba leaves extract (Ginko capsules®; Arab company for pharmaceutical and medicinal plants, MEPACO-MEDIFOOD, Egypt) were used in the current study in doses of 50 and 100 mg/kg/day, p. o. [39, 40]. All other chemicals were of the highest available commercial grade.

Ovariectomy

Rats were anesthetized with thiopental sodium (20 mg/kg; i. p.) and then ovariectomized according to the method described by Turner et al. (2000) [38].

Cerebral ischemia induction

Two months after the operation, animals were starved for 12 h before surgery and then anesthetized with thiopental (20 mg/kg; i. p.). A longitudinal cervical incision (2 cm) was made lateral to the midline and the common carotid artery (CCA) was carefully dissected. Ischemia was induced by placing nontraumatic microvascular clip on left CCA just prior to its bifurcation [41]. During ischemia, rats were monitored for body temperature constant at 36.5±0.5˚C using a heating pad and respiration pattern. The vascular occlusion was maintained for 30 min, and then the clips were removed to resume blood flow to the ischemic region for 24 h [42]. Finally, the incisions were sutured, the animal was allowed to recover from anesthesia, and returned to a warm cage for recuperation during reperfusion period for 24 h.

Experimental design

Rats were randomly allocated into 5 groups (8-12 rats each):

Group I: Sham-operated rats.

Group II: Ovariectomized rats [OVX] that received one ml tab water daily one month after ovariectomization and continued for another month.

Group III: Ovariectomized rats that weren't treated and underwent cerebral ischemia-reperfusion injury [OVX+I/R] 2 mo after ovariectomization.

Group IV: Ovariectomized rats treated one month after ovariectomization with Ginkgo biloba leaves extract (50 mg/kg/day; p. o.). Treatment continued for another month followed by cerebral ischemia-reperfusion injury [(OVX+I/R)+Gin50].

Group V: Ovariectomized rats treated one month after ovariectomization with Ginkgo biloba leaves extract (100 mg/kg/day; p. o.). Treatment continued for another month followed by cerebral ischemia-reperfusion injury [(OVX+I/R)+Gin100].

Biochemistry

At the end of the experiment, blood samples were withdrawn from the retro-orbital venous plexus of 18 h food-deprived rats. Collected blood samples were allowed to stand for 10 min at room temperature then centrifuged at 4oC using cooling centrifuge (Laborezentrifugen, 2k15, Sigma, Germany) at 3000 r. p. m for 10 min and sera were separated for the assessment of serum level of malondialdehyde (MDA), Nitric oxide (NO) metabolites, tumor necrosis factor alpha(TNF-α) expression and reduced glutathione (GSH) according to the methods adopted by Ruiz-Larrea et al. (1994)[43], Miranda et al.(2001)[44], Brouckaert et al.(1993)[45] and Ellman (1959) [46]and modified by Bulaj et al. (1998)[47] respectively using commercially available kits (Biodiagnostic, Egypt).

Tissue sampling

Directly after collecting the last blood sample in the experiment, rats were decapitated under light diethyl ether–anesthesia and their brain were carefully isolated and dissected through the midline into two hemispheres. 0.5g of the affected hemisphere was homogenized (using MPW–120 homogenizer, Med instruments, Poland); the homogenate was centrifuged using a cooling centrifuge (Laborezentrifugen, 2k15, Sigma, Germany) at 3000 r. p. m for 10 min; the supernatant was taken for the determination of brain level of MDA, NO metabolites, TNF-α expression and GSH according to the methods adopted by Ruiz-Larrea et al. (1994)[43], Miranda et al.(2001)[44], Brouckaert et al.(1993)[45] and Ellman (1959)[46] and modified by Bulaj et al.(1998) [47] respectively using commercially available kits (Biodiagnostic, Egypt).

Histopathological examination

Brain tissues from different groups were immediately fixed in 10% neutral buffered formalin and embedded in paraffin wax. 4 µm thick sections were stained with Hematoxylin and Eosin (H & E) and examined using binocular Olympus CX31 microscope [48].

Statistical analysis

All the values are presented as means±standard error of the means (SE). Comparisons between different groups were carried out using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc tests. The difference was considered significant when p˂0.05. GraphPad prism®software (version 6.00) was used to carry out these statistical tests.

RESULTS

Effect of Ginkgo biloba leaves extract on serum biochemical parameters

Cerebral I/R injury in ovariectomized rats resulted in a significant elevation in serum concentration of MDA, NO metabolites and TNF-α and a significant decrease in serum concentration of GSH when compared to either sham-operated or ovariectomized groups. Administration of Ginkgo biloba leaves extract (50 and 100 mg/Kg/day; p. o.) improved all the aforementioned parameters, and the effect of high dose was better than the low dose. Ginkgo biloba leaves extract (50 mg/Kg/day; p. o.) managed to decrease the elevated serum concentration of MDA, NO metabolites and TNF-α to 71%, 82% and 67% respectively when compared to ovariectomized rats undergoing cerebral I/R injury.

Moreover, Ginkgo biloba leaves extract (50 mg/Kg/day; p. o.) succeeded to elevate the diminished serum GSH concentration to156 % when compared to ovariectomized rats undergoing cerebral I/R injury. While Ginkgo biloba leaves extract (100 mg/Kg/day; p. o.) managed to decrease the elevated serum concentration of MDA, NO metabolites and TNF-α to 66%, 61% and 24% respectively when compared to ovariectomized rats undergoing cerebral I/R injury. Moreover, Ginkgo biloba leaves extract (100 mg/Kg/day; p. o.) succeeded to normalize the diminished serum GSH concentration and elevated it to 252 % when compared to ovariectomized rats undergoing cerebral I/R injury (table 1).

Table 1: Effect of Ginkgo biloba leaves extract (50 and 100 mg/Kg/day; p. o.) on serum concentration of GSH, MDA, NO metabolites and TNF-α after cerebral ischemia-reperfusion injury in ovariectomized rats

Groups Serum parameters
GSH (µmol/ml)
Sham-operated 22.02±1.76
OVX 22.18±1.75
(OVX+I/R) 9.41a,b±0.61
(OVX+I/R)+Gin50 14.71±0.57
(OVX+I/R)+Gin100 23.68c±2.73

The administration of drugs was started one month after ovariectomization and continued for another month, followed by cerebral ischemia-reperfusion injury, Data is presented as mean±SE (n=10). aSignificantly different from the sham-operated group at p<0.05 (Tukey’s post hoc test). bSignificantly different from OVX group at p<0.05 (Tukey’s post hoc test). cSignificantly different from OVX+I/R group at p<0.05 (Tukey’s post hoc test).

Table 2: Effect of Ginkgo biloba leaves extract (50 and 100 mg/Kg/day; p. o.) on brain tissue concentration of GSH, MDA, NO metabolites and TNF-α after cerebral ischemia-reperfusion injury in ovariectomized rats

Groups Brain tissue parameters
GSH (µmol/g tissue)
Sham-operated 6.28±0.31
OVX 5.65a±0.44
(OVX+I/R) 4.00b±0.03
(OVX+I/R)+Gin50 4.50a±0.05
(OVX+I/R)+Gin100 6.27c±0.42

The administration of drugs was started one month after ovariectomization and continued for another month, followed by cerebral ischemia-reperfusion injury, Data is presented as mean±SE (n=10). aSignificantly different from the sham-operated group at p<0.05 (Tukey’s post hoc test). bSignificantly different from OVX group at p<0.05 (Tukey’s post hoc test). cSignificantly different from OVX+I/R group at p<0.05 (Tukey’s post hoc test).

Histopathological examination of brain tissue

Fig. 1: Photomicrographs of brain sections (H & E X 200); prepared from (A-B) a sham-operated rat showing no histopathological alteration and the normal histological structure of the meninges, cerebral cortex, hippocampus, striatum, and cerebellum were recorded. (C-D) an ovariectomized rat is showing minimal alteration in the overall histopathological picture of the brain. The normal histological structure of the meninges, cerebral cortex, hippocampus, striatum and cerebellum was observed. (E-G) an ovariectomized rat where cerebral I/R injury has been experimentally induced showing degeneration and nuclear pyknosis in the neurons of the cerebral cortex, and in the hippocampus. Focal eosinophilic plagues formation was clearly observed in the striatum. (H-J) an ovariectomized rat with cerebral I/R injury and treated with Ginkgo biloba leaves extract (50 mg/Kg/day; p. o.) showing moderate degeneration and nuclear pyknosis in some neurons of the cerebral cortex, as well as the hippocampus. Focal gliosis was detected in the cerebrum. (I-K) an ovariectomized rat with cerebral I/R injury and treated with Ginkgo biloba leaves extract (100 mg/Kg/day; p. o.) showing an improved overall histopathological features where the hippocampal neurons showed normal histological structure, while the cerebral cortex showed minimal neuronal pyknosis

Effect of Ginkgo biloba leaves extract on brain tissue parameters

Cerebral I/R injury after ovariectomization resulted in a significant elevation in brain tissue concentration of MDA, NO metabolites and TNF-α and a significant decrease in brain tissue concentration of GSH when compared to either sham-operated or ovariectomized groups. Administration of Ginkgo biloba leaves extract (50 and 100 mg/Kg/day; p. o.) improved all the aforementioned parameters and the effect of high dose was better than the low dose. Ginkgo biloba leaves extract (50 mg/Kg/day; p. o.) decreased the elevated brain tissue concentration of MDA, NO metabolites and TNF-α to 87%, 79% and 74% respectively when compared to ovariectomized rats undergoing cerebral I/R injury. Moreover, Ginkgo biloba leaves extract (50 mg/Kg/day; p. o.) elevated the diminished brain tissue GSH concentration to 113% when compared to ovariectomized rats undergoing cerebral I/R injury.

While, Ginkgo biloba leaves extract (100 mg/Kg/day; p. o.) normalized the diminished brain tissue GSH concentration and decreased the elevated brain tissue concentration of MDA, NO metabolites and TNF-α to 77%, 56% and 52% respectively when compared to ovariectomized rats undergoing cerebral I/R injury. Ginkgo biloba leaves extract (100 mg/Kg/day; p. o.) succeeded to normalize the diminished serum GSH concentration and elevated it to 157% when compared to ovariectomized rats undergoing cerebral I/R injury (table 2).

DISCUSSION

In the present study, ovariectomization of rats resulted in the elevation of serum and brain tissue concentration of anti-inflammatory parameters as TNF-α; oxidative stress parameters as MDA and NO metabolites and a decrease in antioxidant parameters as GSH. These changes are attributable to the loss of the estrogen and its evident neuroprotection. Estrogen exerts its neuroprotective actions through different mechanisms; the antioxidant free-radical scavenging pathway being the most important [49]. The formation of ROS in cases of estrogen depletion plays a major role in the ischemic damage [50]. Arslan et al. (2011) stated that that ovariectomization in rats lead to an elevation in plasma concentrations of MDA and NO metabolites [51]. Also, Ozacmak and Sayan (2009) added that ovariectomization in rats lead to a decline in GSH concentrations along with the elevation in MDA concentrations in the striatum, hippocampus, and cortex when compared to normal rats [50]. Moreover, ovariectomization caused a minimal change in the histological structure of the brain tissue, and the overall histopathological picture of the ovariectomized group was normal when compared to the sham-operated group. This data is in agreement with Aly et al. (2011) who demonstrated that ovariectomized rats showed normal histopathological features [52].

The present study showed that cerebral I/R injury induced deleterious biochemical changes in serum as well as in the brain tissue and caused substantial brain injury. In ovariectomized rats undergoing cerebral I/R injury; serum and brain tissue concentration of inflammatory parameters as TNF-α; oxidative stress parameters as MDA and NO metabolites were significantly elevated whereas serum and brain tissue concentration of antioxidant parameters as GSH was significantly decreased when compared to ovariectomized group. The histopathological picture of ovariectomized rats undergoing cerebral I/R injury showed degeneration and nuclear pyknosis in the neurons of the cerebral cortex and in the hippocampus. Focal eosinophilic plagues formation was clearly observed in the striatum. Although significant cerebral damage occurs during an ischemic episode, further brain damage can occur after blood flow restoration [53].

Previous studies reported that cerebral I/R injury caused deleterious events in young and adult rats [41, 54]. One possible mechanism for brain damage that occurs during reperfusion involves generation of ROS [55]. Reduced glutathione (GSH) is an endogenous antioxidant scavenging free radicals and protecting against ROS and oxidative stress. Preserving GSH-mediated antioxidant defense is critical for cell survival [56]. Damage to membrane lipids, specifically lipolysis during ischemia and free radical-mediated peroxidation of polyunsaturated fatty acids during reperfusion [57] and MDA production; which is the end product of lipid peroxidation, as well as significant increase in its concentration in brain tissue leads to many of the outcomes observed in cerebral I/R injury [41]. Ozacmak and Sayan reported that MDA concentration in brain tissue samples increases significantly in cerebral I/R injury accompanied by a significant decrease in GSH levels in the hippocampus when compared to the ovariectomized group [50].

The present study demonstrated that serum and brain levels of NO metabolites were significantly elevated in cerebral I/R injury group when compared to ovariectomized group. The formation of NO metabolites increase in brain injury, where the L-arginine-NO pathway is activated by the increased levels of cytokines and endotoxins [42]. Morikawa et al.(1992) and Yamamoto et al.(1992) demonstrated a significant increase in NO production by cerebral vascular endothelial cells, which might have exerted a neuroprotective effect in focal cerebral ischemia by increasing blood flow to marginally ischemic tissues [58, 59].

It has been well established that during brain ischemia, pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, are vastly produced by different activated cell types as endothelial cells, microglia, astrocytes, and neurons [60]. Hindering the production of these pro-inflammatory cytokines would be an important approach to protecting against ischemic brain injury. In the present study, we observed that TNF-α expression was significantly elevated in cerebral I/R injury group when compared to ovariectomized group. This data is in agreement with Brown et al. (2010) who stated that a series of pro-inflammatory cytokines and chemokines are induced in cerebral injury [3]. Jin et al. (2015) demonstrated that TNF-α was highly expressed in ischemic brain injury in rats and was significantly reduced after treatment [61]. Silva et al. (2015) also observed marked cytokine and chemokine up-regulation (IL-1 and TNF-α) after cerebral I/R injury in mice [62].

Administration of Ginkgo biloba leaves extract (50 mg/Kg/day; p. o.) significantly decreased the elevated serum and brain tissue concentrations of TNF-α, MDA and NO metabolites. It also increased serum and brain tissue concentration of GSH when compared to ovariectomized cerebral I/R injury group. The histopathological picture of rats treated with Ginkgo biloba leaves extract (50 mg/Kg/day; p. o.) showed moderate degeneration and nuclear pyknosis in some neurons of the cerebral cortex as well as the hippocampus. Focal gliosis was also detected.

Ginkgo biloba leaves extract (100 mg/Kg/day; p. o.) improved and almost normalized all the biochemical parameters measured in sera and brain tissues. Moreover, an improvement in the overall histopathological features was noted in rats treated with Ginkgo biloba leaves extract (100 mg/Kg/day; p. o.); where the hippocampal neurons showed the normal histological structure and the cerebral cortex showed minimal neuronal pyknosis. It was clear that the effects of the high dose were better than the low dose.

Previous preclinical studies support the hypothesis that Ginkgo biloba may be effective in the treatment and prevention of age-related neurodegenerative disorders [27]. Many studies have proposed that the beneficial action of Ginkgo biloba leaves extract was mainly due to its free-radical scavenging effect [29]. Wei et al.(2000) demonstrated that pretreating cerebellar granule cells with Ginkgo biloba effectively caused attenuation of oxidative damage triggered by hydrogen peroxide/ferrous sulphate (H2O2/FeSO4) [63]. In another study, Ginkgo biloba was found to be able to attenuate the basal as well as the induced levels of H2O2-related ROS [60]. In addition to the direct reduction of ROS, Ginkgo biloba also stabilized the cellular redox state by up-regulation of activity of antioxidant enzymes [30]. Ginkgo biloba increased the activity of superoxide dismutase and catalase in rat hippocampus [64]. Ginkgo biloba also enhanced the activity of two enzymes essential for reduction and synthesis of GSH, which are glutathione reductase and gamma-glutamylcysteinyl synthetase [31]. The flavonoid fraction of Ginkgo biloba is suggested to be mainly responsible for its antioxidant properties [31]. Moreover, Ginkgo biloba has been proved to have anti-inflammatory effects. These effects may be due to the combined actions of its contents: ginkgolide and flavonoid [33]. Ginkgo biloba inhibits the production of pro-inflammatory cytokines TNF-α and IL-1 in rats [65]. Other mechanisms may also be involved in neuroprotective actions of Ginkgo biloba including its anti-apoptotic effect, protection against mitochondrial dysfunction, amyloidogenesis, and induction of growth factors [27].

CONCLUSION

In conclusion, the current study revealed that cerebral ischemia-reperfusion injury induced severe deleterious serum and brain tissue biochemical changes as well as brain histopathological changes in ovariectomized rats lacking the neuroprotective features of estrogen. Ginkgo biloba leaves extract given in two doses (50 and 100 mg/kg/day, p. o.) for one month before induction of cerebral I/R injury in ovariectomized rats markedly decreased the elevated serum and brain tissue concentration of inflammatory parameters as TNF-α and serum and brain tissue concentration of oxidative stress parameters as MDA and NO metabolites. On the other hand, serum and brain tissue concentration of antioxidant parameters as GSH was significantly elevated in rats treated with Ginkgo biloba. Ginkgo biloba also improved the overall histopathological pictures of rats when compared to ovariectomized cerebral I/R injury group. Thus the study suggests Ginkgo biloba as a potent protective agent against cerebral I/R injury in ovariectomized rats. This may give an insight to protection against cerebral I/R injury effects in estrogen deficient females either due to reproductive senescence or depletion of endogenous estrogens. The proposed neuroprotective mechanisms of action of Ginkgo biloba include its anti-inflammatory and anti-oxidant actions.

ABBREVIATION

Ischemia-reperfusion (I/R), 17b-estradiol (E2), Estrogen replacement therapy (ERT), Reactive oxygen species (ROS), Common carotid artery (CCA), Malondialdehyde (MDA), Nitric oxide (NO), Tumor necrosis factor alpha(TNF-α), Reduced glutathione (GSH).

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. McCullough LD, Hurn PD. Estrogen and ischemic neuroprotection: an integrated view. Trends Endocrinol Metab 2003;14:228-35.
  2. Viscoli CM, Brass LM, Kernan WN, Sarrel PM, Suissa S, Horwitz RI. A clinical trial of estrogen-replacement therapy after ischemic stroke. N Engl J Med 2001;345:1243-9.
  3. Brown CM, Mulcahey TA, Filipek NC, Wise PM. Production of proinflammatory cytokines and chemokines during neuroinflammation: novel roles for estrogen receptors alpha and beta. Endocrinology 2010;151:4916-25.
  4. Franceschi C, Bonafe M, Valensin S, Olivieri F, De Luca M, Ottaviani E, et al. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 2000;908:244-54.
  5. Salem ML. Estrogen, a double-edged sword: modulation of TH1-and TH2-mediated inflammations by differential regulation of TH1/TH2 cytokine production. Curr Drug Targets: Inflammation Allergy 2004;3:97-104.
  6. Abu-Taha M, Rius C, Hermenegildo C, Noguera I, Cerda-Nicolas JM, Issekutz AC, et al. Menopause and ovariectomy cause a low grade of systemic inflammation that may be prevented by chronic treatment with low doses of estrogen or losartan. J Immunol 2009;183:1393-402.
  7. Salvioli S, Capri M, Valensin S, Tieri P, Monti D, Ottaviani E, et al. Inflamm-aging, cytokines and aging: state of the art, new hypotheses on the role of mitochondria and new perspectives from systems biology. Curr Pharm Des 2006;12:3161-71.
  8. Vegeto E, Benedusi V, Maggi A. Estrogen anti-inflammatory activity in brain: a therapeutic opportunity for menopause and neurodegenerative diseases. Front Neuroendocrinol 2008;29:507-19.
  9. Suzuki S, Brown CM, Wise PM. Neuroprotective effects of estrogens following ischemic stroke. Front Neuroendocrinol 2009;30:201-11.
  10. de Rivero Vaccari JP, Patel HH, Brand FJ, Perez-Pinzon MA, Bramlett HM, Raval AP. Estrogen receptor beta signaling alters cellular inflammasomes activity after global cerebral ischemia in reproductively senescence female rats. J Neurochem 2016;136:492-6.
  11. Grodstein F, Stampfer MJ, Colditz GA, Willett WC, Manson JE, Joffe M, et al. Postmenopausal hormone therapy and mortality. N Engl J Med 1997;336:1769-75.
  12. Liao SL, Chen WY, Kuo JS, Chen CJ. Association of serum estrogen level and ischemic neuroprotection in female rats. Neurosci Lett 2001;297:159-62.
  13. Straub RH. The complex role of estrogens in inflammation. Endocr Rev 2007;28:521-74.
  14. Waters EM, Yildirim M, Janssen WG, Lou WY, McEwen BS, Morrison JH, et al. Estrogen and aging affect the synaptic distribution of estrogen receptor beta immunoreactivity in the CA1 region of female rat hippocampus. Brain Res 2011;1379:86-97.
  15. Schaller B, Graf R. Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy. J Cereb Blood Flow Metab 2004;24:351-71.
  16. Aronowski J, Strong R, Grotta JC. Reperfusion injury: demonstration of brain damage produced by reperfusion after transient focal ischemia in rats. J Cereb Blood Flow Metab 1997;17:1048-56.
  17. Kuroda S, Siesjo BK. Reperfusion damage following focal ischemia: pathophysiology and therapeutic windows. Clin Neurosci 1997;4:199-212.
  18. Pan J, Konstas AA, Bateman B, Ortolano GA, Pile-Spellman J. Reperfusion injury following cerebral ischemia: pathophysiology, MR imaging, and potential therapies. Neuroradiology 2007;49:93-102.
  19. Huh H, Staba EJ. The botany and chemistry of Ginkgo biloba L. J Herbs Spices Med Plants 1992;1:91-124.
  20. Puttalingamma V. Ginkgo biloba “living fossil”, wonderful medicinal plant-A review. Int J Adv Res 2015;3:506-11.
  21. Le Bars PL. The magnitude of effect and special approach to ginkgo biloba extract EGb 761 in cognitive disorders. Pharmacopsychiatry 2003;36 Suppl 1:S44-9.
  22. Smith PF, Maclennan K, Darlington CL. The neuroprotective properties of the Ginkgo biloba leaf: a review of the possible relationship to platelet-activating factor (PAF). J Ethnopharmacol 1996;50:131-9.
  23. Shi C, Zhao L, Zhu B, Li Q, Yew DT, Yao Z, et al. Protective effects of Ginkgo biloba extract (EGb761) and its constituents quercetin and ginkgolide B against beta-amyloid peptide-induced toxicity in SH-SY5Y cells. Chem Biol Interact 2009;181:115-23.
  24. Smith JV, Luo Y. Elevation of oxidative free radicals in Alzheimer's disease models can be attenuated by Ginkgo biloba extract EGb 761. J Alzheimers Dis 2003;5:287-300.
  25. Luo Y. Ginkgo biloba neuroprotection: therapeutic implications in Alzheimer's disease. J Alzheimers Dis 2001;3:401-7.
  26. Holstein N. Ginkgo special extract EGb 761 in tinnitus therapy. An overview of results of completed clinical trials. Fortschr Med Orig 2001;118:157-64.
  27. Shi C, Liu J, Wu F, Yew DT. Ginkgo biloba extract in Alzheimer's disease: from action mechanisms to medical practice. Int J Mol Sci 2010;11:107-23.
  28. Dua JS, Prasad DN, Tripathi AC, Gupta R. Role of traditional medicine in neuropsychopharmacology. Asian J Pharm Clin Res 2009;2:72-6.
  29. Smith JV, Luo Y. Studies on molecular mechanisms of Ginkgo biloba extract. Appl Microbiol Biotechnol 2004;64:465-72.
  30. Ahlemeyer B, Krieglstein J. Neuroprotective effects of Ginkgo biloba extract. Cell Mol Life Sci 2003;60:1779-92.
  31. Sasaki K, Hatta S, Wada K, Ueda N, Yoshimura T, Endo T, et al. Effects of an extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver. Life Sci 2002;70:1657-67.
  32. Braquet P, Hosford D. Ethnopharmacology and the development of natural PAF antagonists as therapeutic agents. J Ethnopharmacol 1991;32:135-9.
  33. Chan PC, Xia Q, Fu PP. Ginkgo biloba leave extract: biological, medicinal, and toxicological effects. J Environ Sci Health Part C: Environ Carcinog Ecotoxicol Rev 2007;25:211-44.
  34. Shi C, Xiao S, Liu J, Guo K, Wu F, Yew DT, et al. Ginkgo biloba extract EGb761 protects against aging-associated mitochondrial dysfunction in platelets and hippocampi of SAMP8 mice. Platelets 2010;21:373-9.
  35. Shi C, Fang L, Yew DT, Yao Z, Xu J. Ginkgo biloba extract EGb761 protects against mitochondrial dysfunction in platelets and hippocampi in ovariectomized rats. Platelets 2010;21:53-9.
  36. Berrocal M, Marcos D, Sepulveda MR, Perez M, Avila J, Mata AM. Altered Ca2+dependence of synaptosomal plasma membrane Ca2+-ATPase in the human brain affected by Alzheimer's disease. FASEB J 2009;23:1826-34.
  37. Institute of Laboratory Animal Resources. Committee on Care Use of Laboratory Animals; 2010.
  38. National Institutes of Health. Division of research resources guide for the care and use of laboratory animals. National Academies; NIH Publication No. 85-23, U. S. Department of Health, Education and Welfare; 1985.
  39. Turner RT, Maran A, Lotinun S, Hefferan T, Evans GL, Zhang M, et al. Animal models for osteoporosis. Rev Endocr Metab Disord 2001;2:117-27.
  40. Lee EJ, Chen HY, Wu TS, Chen TY, Ayoub IA, Maynard KI. Acute administration of Ginkgo biloba extract (EGb 761) affords neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rats. J Neurosci Res 2002;68:636-45.
  41. Chandrasekaran K, Mehrabian Z, Spinnewyn B, Chinopoulos C, Drieu K, Fiskum G. Neuroprotective effects of bilobalide, a component of Ginkgo biloba extract (EGb 761) in global brain ischemia and in excitotoxicity-induced neuronal death. Pharmacopsychiatry 2003;36 Suppl 1:S89-94.
  42. Renolleau S, Aggoun-Zouaoui D, Ben-Ari Y, Charriaut-Marlangue C. A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis. Stroke 1998;29:1454-61.
  43. Kuluz JW, Prado RJ, Dietrich WD, Schleien CL, Watson BD. The effect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats. Stroke 1993;24:2023-9.
  44. Ruiz-Larrea MB, Leal AM, Liza M, Lacort M, de Groot H. Antioxidant effects of estradiol and 2-hydroxyestradiol on iron-induced lipid peroxidation of rat liver microsomes. Steroids 1994;59:383-8.
  45. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001;5:62-71.
  46. Brouckaert P, Libert C, Everaerdt B, Takahashi N, Cauwels A, Fiers W. Tumor necrosis factor, its receptors and the connection with interleukin 1 and interleukin 6. Immunobiology 1993;187:317-29.
  47. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.
  48. Bulaj G, Kortemme T, Goldenberg DP. Ionization-reactivity relationships for cysteine thiols in polypeptides. Biochemistry 1998;37:8965-72.
  49. Bancroft J, Stevens A, Turner D. Theory and practice of histological techniques: Churchill Livingstone New York. the text; 1996. p. 766.
  50. Garcia-Segura LM, Azcoitia I, Don Carlos LL. Neuroprotection by estradiol. Prog Neurobiol 2001;63:29-60.
  51. Ozacmak VH, Sayan B. The Effects of 17[beta] Estradiol, 17[alpha] Estradiol and progesterone on oxidative stress biomarkers in ovariectomized female rat brain subjected to global cerebral ischemia. Physiol Res 2009;58:909-12.
  52. Arslan A, Orkun S, Aydin G, Keles I, Tosun A, Arslan M, et al. Effects of ovariectomy and ascorbic acid supplement on oxidative stress parameters and bone mineral density in rats. Libyan J Med 2011;6:10.
  53. Aly HF, Metwally FM, Ahmed HH. Neuroprotective effects of dehydroepiandrosterone (DHEA) in rat model of Alzheimer's disease. Acta Biochim Pol 2011;58:513-20.
  54. Pulsinelli WA, Brierley JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 1982;11:491-8.
  55. Fellman V, Raivio KO. Reperfusion injury as the mechanism of brain damage after perinatal asphyxia. Pediatr Res 1997;41:599-606.
  56. Traystman RJ, Kirsch JR, Koehler RC. Oxygen radical mechanisms of brain injury following ischemia and reperfusion. J Appl Physiol 1991;71:1185-95.
  57. Anderson MF, Nilsson M, Eriksson PS, Sims NR. Glutathione monoethyl ester provides neuroprotection in a rat model of stroke. Neurosci Lett 2004;354:163-5.
  58. White BC, Sullivan JM, DeGracia DJ, O'Neil BJ, Neumar RW, Grossman LI, et al. Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. J Neurol Sci 2000;179(Suppl 1-2):1-33.
  59. Morikawa E, Huang Z, Moskowitz MA. L-arginine decreases infarct size caused by middle cerebral arterial occlusion in SHR. Am J Physiol 1992;263:H1632-5.
  60. Yamamoto S, Golanov EV, Berger SB, Reis DJ. Inhibition of nitric oxide synthesis increases focal ischemic infarction in rat. J Cereb Blood Flow Metab 1992;12:717-26.
  61. Wu Y, Wu Z, Butko P, Christen Y, Lambert MP, Klein WL, et al. Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic caenorhabditis elegans. J Neurosci 2006; 26:13102-13.
  62. Jin Z, Liang J, Wang J, Kolattukudy PE. MCP-induced protein 1 mediates the minocycline-induced neuroprotection against cerebral ischemia/reperfusion injury in vitro and in vivo. J Neuroinflammation 2015;12:39.
  63. Silva B, Sousa L, Miranda A, Vasconcelos A, Reis H, Barcelos L, et al. Memory deficit associated with increased brain proinflammatory cytokine levels and neurodegeneration in acute ischemic stroke. Arq Neuropsiquiatr 2015;73:655-9.
  64. Wei T, Ni Y, Hou J, Chen C, Zhao B, Xin W. Hydrogen peroxide-induced oxidative damage and apoptosis in cerebellar granule cells: protection by Ginkgo biloba extract. Pharmacol Res 2000;41:427-33.
  65. Bridi R, Crossetti FP, Steffen VM, Henriques AT. The antioxidant activity of standardized extract of Ginkgo biloba (EGb 761) in rats. Phytother Res 2001;15:449-51.
  66. Maclennan KM, Darlington CL, Smith PF. The CNS effects of Ginkgo biloba extracts and ginkgolide B. Prog Neurobiol 2002;67:235-57.

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