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EDITOR-IN-CHIEF DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA

[email protected] EDITORS PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain [email protected] PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy [email protected] PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China [email protected] PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan [email protected] PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia [email protected] PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA [email protected] PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA [email protected] PROFESSOR YASUHIRO TEZUKA Faculty of Pharmaceutical Sciences Hokuriku University Ho-3 Kanagawa-machi, Kanazawa 920-1181, Japan [email protected] PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK [email protected]

HONORARY EDITOR PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences, University of Portsmouth, Portsmouth, PO1 2DT U.K. [email protected]

ADVISORY BOARD Prof. Viqar Uddin Ahmad Karachi, Pakistan Prof. Giovanni Appendino Novara, Italy Prof. Yoshinori Asakawa Tokushima, Japan Prof. Roberto G. S. Berlinck São Carlos, Brazil Prof. Anna R. Bilia Florence, Italy Prof. Maurizio Bruno Palermo, Italy Prof. César A. N. Catalán Tucumán, Argentina Prof. Josep Coll Barcelona, Spain Prof. Geoffrey Cordell Chicago, IL, USA Prof. Fatih Demirci Eskişehir, Turkey Prof. Dominique Guillaume Reims, France Prof. Ana Cristina Figueiredo Lisbon, Portugal Prof. Cristina Gracia-Viguera Murcia, Spain Prof. Duvvuru Gunasekar Tirupati, India Prof. Hisahiro Hagiwara Niigata, Japan Prof. Kurt Hostettmann Lausanne, Switzerland Prof. Martin A. Iglesias Arteaga Mexico, D. F, Mexico Prof. Leopold Jirovetz Vienna, Austria Prof. Vladimir I Kalinin Vladivostok, Russia Prof. Niel A. Koorbanally Durban, South Africa

Prof. Chiaki Kuroda Tokyo, Japan Prof. Hartmut Laatsch Gottingen, Germany Prof. Marie Lacaille-Dubois Dijon, France Prof. Shoei-Sheng Lee Taipei, Taiwan Prof. Imre Mathe Szeged, Hungary Prof. Ermino Murano Trieste, Italy Prof. M. Soledade C. Pedras Saskatoon, Canada Prof. Luc Pieters Antwerp, Belgium Prof. Peter Proksch Düsseldorf, Germany Prof. Phila Raharivelomanana Tahiti, French Polynesia Prof. Luca Rastrelli Fisciano, Italy Prof. Stefano Serra Milano, Italy Prof. Monique Simmonds Richmond, UK Dr. Bikram Singh Palampur, India Prof. John L. Sorensen Manitoba, Canada Prof. Johannes van Staden Scottsville, South Africa Prof. Valentin Stonik Vladivostok, Russia Prof. Winston F. Tinto Barbados, West Indies Prof. Sylvia Urban Melbourne, Australia Prof. Karen Valant-Vetschera Vienna, Austria

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Natural Product Communications

Advances in Herbal Medicine for Treatment of Ischemic Brain Injury

2014 Vol. 9 No. 7 1045 - 1055

Nilanjan Ghosha, Rituparna Ghosha, Zulfiqar A Bhatb, Vivekananda Mandalc, Sitesh C. Bachard, Namsa D. Nimae, Otimenyin O. Sundayf and Subhash C. Mandalg a

Dr B.C. Roy College of Pharmacy and Allied Health Sciences, Durgapur, India 713206 Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, India 190006 c Institute of Pharmacy, Guru Ghasidas University, Bilaspur, India, 495009 d Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Dhaka, Bangladesh e Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, India 784028 f Department of Pharmacology, Faculty of Pharmaceutical Sciences, University of Jos, Jos, Plateau State, Nigeria g Pharmacognosy and Phytotherapy Research Laboratory, Division of Pharmacognosy, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India 700032 b

[email protected] Received: February 17th, 2014; Accepted: May 26th, 2014

Ischemic brain injury is one of the leading causes of death worldwide and has attracted a lot of attention in the field of drug discovery. Cerebral ischemia is a complex pathological process involving a series of mechanisms, including generation of free radicals, oxidative stress, disruption of the membrane function, release of neurotransmitters and apoptosis. Thrombolytic therapy is the most effective therapeutic strategy, but the benefits are far from being absolute. Increased attention in the field of drug discovery has been focused on using natural compounds from traditional medicinal herbs for neuroprotection, which appears to be a promising therapeutic option for cerebral ischemia with minimal systemic adverse effects that could limit their long term use. The scenario calls for extensive investigations which can result in the development of lead molecules for neuroprotection in the future. In this context, the present review focuses on possible mechanisms underlying the beneficial effects of herbal drugs in patients with cerebral ischemic injury. Natural compounds have been demonstrated to have neurofunctional regulatory actions with antioxidative, anti-inflammatory, calcium antagonizing and anti-apoptotic activities. Among the several leads obtained from plant sources as potential neuroprotective agents, resveratrol, EGb761, curcumin and epigallocatechin-3-gallate have shown significant therapeutic benefits in cerebral ischemic conditions. However, ligustilide, tanshinone, scutellarin and shikonin are the few lead molecules which are under investigation for treatment of cerebral ischemia. Keywords: Oxidative stress, Neurodegeneration, Cerebral ischemia, Apoptosis, Blood brain barrier, Herbal Drugs.

Stroke is the world's second leading cause of mortality, with a lifetime risk of around 10% [1]. It is a condition of acute neurologic dysfunction generally resulting from an ischemic brain injury characterized by the sudden occurrence of symptoms and signs corresponding to the involvement of focal areas of the brain. The two main types of stroke are the ischemic and the hemorrhagic, accounting for approximately 85% and 15% of cases, respectively [2]. When an ischemic stroke occurs, the blood supply to the brain is interrupted, and brain cells are deprived of glucose and oxygen. The most frequent causes of such focal obstructions of blood flow within the brain are embolisms. Ischemia is, in fact, defined as a reduction in blood flow sufficient to alter normal cellular functions. Neurons are very sensitive to ischemia and even brief periods of exposure to this dysfunction can initiate a complex sequence of events leading to the loss of neurons. The CA1 pyramidal neurons of the hippocampus are highly susceptible to ischemia. Ischemic stroke is very complex with multiple etiologies and variable clinical symptoms [3]. The preferred treatment to limit brain injury that follows stroke is the early reperfusion of the ischemic brain, while thrombolytic therapy has decreased mortality following stroke in patients with acute ischemic stroke [4]. Nevertheless, tissue damage often results from both the transient ischemic insult and the reperfusion process given that the latter induces an inflammatory response that causes additional injury to the cerebral microcirculation and adjacent brain tissue. Reperfusion often causes generation of intracellular reactive oxygen species (ROS), calcium overload and excitotoxic cell injury and inflammation, which

ultimately lead to irreversible brain injury [5]. This review considers the pathogenesis of ischemic stroke and focuses on the pharmacological mechanisms underlying neuroprotective mechanisms of traditional medicinal herbs that demonstrate protective effects on ischemic brain injury. Ischemic stroke pathophysiology: The pathophysiology of stroke is complex and involves many processes, including energy failure, loss of cell ion homeostasis, acidosis, increased intracellular calcium levels, excitotoxicity, free radical-mediated toxicity, cytokine-mediated cytotoxicity, disruption of the blood-brain barrier (BBB), activation of glial cells, and infiltration of leukocytes. Within a few minutes of a cerebral ischemia, the core of brain tissue exposed to severe blood flow reduction is injured and subsequently undergoes necrotic cell death [6]. This necrotic core is surrounded by a zone of less severely affected tissue which is turned functionally inactive by ischemia but remains metabolically active. Necrosis is morphologically characterized by initial cellular and organelle swelling, subsequent disruption of nuclear, organelle, and plasma membranes, and disintegration of nuclear structure and cytoplasmic organelles with extrusion of cell contents into the extracellular space [7]. The region bordering the infarct core, known as the ischemic penumbra, comprises as much as half of the total lesion volume during the initial stages of ischemia, and represents the region in which there is opportunity for salvage via post-stroke therapy [8].

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Oxidative stress: Numerous experimental and clinical observations have shown increased free radical formation during ischemic attack, thus indicating involvement of oxidative stress in the pathophysiology of ischemic stroke [9]. ROS play a role in normal physiological processes. However, they are also implicated in a number of disease processes, whereby they mediate damage to cell structures, such as lipids, membranes, proteins, and DNA [10]. Free radicals involved in ischemic brain injury include superoxide anion radical, hydroxyl radical and nitric oxide (NO). The primary source of ROS during ischemic-stroke injury is the mitochondria [10, 11]. Superoxide is generated in the metabolism of arachidonic acid through the cyclooxygenase and lipoxygenase pathways [12]. ROS can also be generated by activated microglia and by infiltration of peripheral leukocytes via the NADPH oxidase system after the reperfusion of ischemic tissue [9]. NO is generated from L-arginine through one of several nitric oxide synthase (NOS) isoforms, including neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). nNOS requires calcium/calmodulin for activation and is expressed by subpopulations of neurons throughout the brain [13]. iNOS is expressed by inflammatory cells such as microglia and monocytes. These two isoforms are mostly damaging to the brain under ischemic conditions. eNOS, on the other hand, has vasodilatory effects and is likely to play a beneficial role by improving local blood flow [13]. NMDA receptor activation has been shown to stimulate NO production by nNOS, and possibly to play a role in excitotoxicmediated injury in ischemic stroke [14]. NO reacts with superoxide to form peroxynitrite (ONOO-) [13]. Both oxygen-derived free radicals and reactive nitrogen species are involved in activating apoptosis and inflammation [10]. Lipid peroxidation appears to play a prominent role in the pathogenesis of stroke and involves generation of an α,β-unsaturated hydroxyalkenal called 4hydroxynonenal (4-HNE). 4-HNE, in turn, covalently modifies membrane transporters such as Na+/K+ ATPase, glucose transporters and glutamate transporters, thereby impairing their function [15]. It has also been shown to trigger toxic pathways as, for instance, the induction of caspase enzymes, the laddering of genomic DNA and the release of cytochrome c from mitochondria, with the eventual outcome of cell death. Inflammation: Brain ischemia-reperfusion-induced inflammatory responses include increased microglial and astrocyte activity, increased production of cytokines, chemokines, adhesion molecules and metalloproteinases, and infiltration of monocytes and leucocytes into injured brain regions [16]. Inflammation is obvious within several hours of ischemia-reperfusion injury. It contributes to secondary damage caused by the microglial activation and resident perivascular and parenchymal macrophages, as well as to infiltration of peripheral inflammatory cells. Endothelial cells, astrocytes and microglia secrete proinflammatory mediators after an ischemic insult [16]. The activation of transcription factors, in turn, results in increased levels of cytokines and increased expression of endothelial cell adhesion molecules (CAMs) in post-stroke brain tissue [17]. A major role in brain inflammation following stroke is attributed to microglia. Once activated, they produce numerous proinflammatory cytokines, as well as toxic metabolites and enzymes [18]. In addition to microglial cells, astrocytes also have an important part in stroke-induced brain inflammation for producing proinflammatory cytokines. There is significant evidence of leukocyte involvement in the pathogenesis of stroke. Leukocytes accumulate in post-ischemic tissues prior to the onset of tissue injury and are believed to mediate reperfusion-induced tissue injury and microvascular dysfunction.

Ghosh et al.

Neutrophils are generally the first leukocyte subtype recruited to the ischemic brain and may potentiate injury by directly secreting injurious substances or other inflammatory mediators [16, 18]. These mediators lead to secondary injury of potentially salvageable tissue within the penumbra surrounding the infarct core. They adhere to endothelial ischemic brain vasculature in the acute phase following stroke and infiltrate into brain parenchyma [16]. Blood Brain Barrier dysfunction: The BBB comprises the endothelial cells that line the capillaries of the brain. The unique characteristics of this barrier include tight intercellular junctions, a complex glycocalyx, lack of pinocytic vesicles, and absence of fenestra. These properties allow for the selective exchange of substances between the systemic circulation and the extracellular fluid compartment of the brain [19]. Both disruptions of the BBB and edema formation play key roles in the development of neurological dysfunction in acute and chronic cerebral ischemia. Animal studies have revealed the molecular cascades that are initiated with ischemia in the cells forming the neurovascular unit and contribute to cell death [19]. Induction of hypoxia inducible factor-1α by loss of oxygen and ATP leads to activation of MMP-2. Additionally, cytokines induce the expression of MMP-3 and MMP-9. Active MMPs degrade the basal lamina and tight junctions of endothelial cells, thus leading to opening of the BBB, which leads to vasogenic edema. COX-2 is inducible and contributes to BBB damage as part of a secondary inflammatory response from 24 to 72 hours after the initial insult [20]. Also, there is considerable evidence that acute ischemic stroke enhances the interactions of the brain endothelium with extravascular CNS cells (astrocytes, microglia, neurons), as well as intravascular cells (platelets, leukocytes), and that these interactions contribute to the injury process [21]. A significant amount of data points towards contribution of NO to ischemia- and inflammation-induced disruption of the BBB [19]. TNFα contributes to the breakdown of the BBB as it enhances the toxicity of NO in brain capillary endothelial cells and involves the generation of superoxide-NO [22]. NO rapidly reacts with superoxide anion to form ONOO− anion [23]. ONOO− is a toxic anion that is capable of generating the highly damaging hydroxyl radical. ONOO− is involved in carrying out nitration of tyrosine which interferes with key enzymes of the tricarboxylic acid cycle, the mitochondrial respiratory chain, mitochondrial Ca2+ metabolism or induced DNA damage, leading to endothelial injury [22]. NOmediated BBB breakdown is predominantly elicited by ONOO− [24]. Glutamate excitotoxicity: Glutamate is one of the major excitatory neurotransmitters in the central nervous system (CNS). It controls various cellular and synaptic functions, cell death and survival, motor functions, learning and memory. In spite of being a physiologically important excitatory neurotransmitter, glutamate plays a pivotal role in various neurological disorders, including ischemic neurological diseases. Its level is increased during cerebral ischemia with excessive neurological stimulation causing the glutamate-induced neuronal toxicity, termed excitotoxicity. This process is considered to be the triggering spark in ischemic neuronal damage [25]. The increase of glutamate results in increased stimulation of AMPARs, KARs and NMDARs on neurons, with consequent influx of Na+ and Ca2+ ions through the channels gated by these receptors. The ion influx is followed by water inflow, resulting in cytotoxic edema. The elevated intracellular calcium leads to mitochondrial dysfunction, activation of proteases and apoptotic mechanisms, accumulation of ROS and release of NO [26].

Herbal medicines for treating ischemic brain injury

The neurotoxicity triggered by NMDA receptors is mediated in large part by activation of nNOS and production of NO and subsequent formation of ONOO-. ONOO- damages DNA, leading to strand breaks and activation of poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a chromatin-associated enzyme, which modifies various nuclear proteins by poly ADP-ribosylation. The modification is dependent on DNA and is involved in the regulation of cellular processes such as differentiation, proliferation and recovery of cells from DNA damage. PARP-1-mediated neuronal death is associated with a decrease in ATP and NAD [23]. After PARP-1 activation, apoptosis-inducing factor (AIF) translocates to the nucleus from mitochondria and triggers chromatin condensation, DNA fragmentation and nuclear shrinkage. This process is followed by cytochrome c release and activation of caspase-3 [26]. Toll like receptors (TLRs): The surface of pathogens typically bear repeating patterns of molecular structure referred to as pathogen-associated molecular patterns (PAMPs). The innate immune system recognizes such pathogens by means of patternrecognition receptors (PRRs) that bind features of these regular patterns [27]. TLRs are transmembrane PRRs that initiate signals in response to diverse PAMPs. TLRs are expressed in different brain cell types such as microglial cells, astrocytes and oligodendrocytes. The ability of TLRs to mediate inflammatory responses in immune cells suggests their involvement in inflammatory responses and ischemia-induced neuronal damage. Expression studies confirm that cerebral ischemia results in the upregulation of mRNA for TLR2, TLR4 and TLR9 in the CNS of mice. TLR4 mutant mice exhibit improved neurological behavior and reduced edema and levels of pro-inflammatory cytokine secretion to the serum such as TNF-α and IL-6 in mouse models of cerebral ischemia [28]. On the other hand, mice that lacked TLR4 had minor expression of strokeinduced interferon regulatory factor 1(IRF-1), iNOS, and COX-2 and mediators implicated in brain damage. In fact, recent findings suggest that, following a stroke, TLRs upregulate the expression of adhesion molecules that facilitate the infiltration of lymphocytes into the ischemic brain region. It also favors the beginning of an inflammatory cascade involving cytokines and chemokines secretion, and NOS and COX-2 expression induction, which contributes to neuronaldamage. TLR activation induces endothelial cells dysfunction, provoking an increase in BBB permeability as activated TLRs induce NFκB activation in glial cells, endothelial cells and infiltrating lymphocytes [29]. The effect on neurons is the activation of the JNK/AP-1 pathway, which contributes to apoptosis and neurodegeneration [30]. Role of herbal drugs in cerebral ischemia A lot of scientific enterprise has indicated that herbal drugs could provide a significant therapeutic breakthrough in the treatment of cerebral ischemic conditions. Some of the compounds that could be of significant therapeutic benefit are discussed in detail in the following section. Resveratrol: Resveratrol (3,4',5-trihydroxystilbene) is a phytoalexin found in the skin and seeds of grapes that has been reported to possess anti-inflammatory, anti-carcinogenic, and antioxidant activities. In addition, this compound has been shown to have significant antioxidant properties in a variety of in vitro and in vivo models. The ability of resveratrol to upregulateheme oxygenase-1 (HO-1) gene expression via antioxidant responsive elements (ARE)-mediated transcriptional activation of NF-E2 related factor-2 (Nrf2) in cultured PC12 cells has also been assessed [31]. A study investigated the oxidative mechanisms underlying the neuroprotective effects of resveratrol in global cerebral ischemia.

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Transient global cerebral ischemia was induced in rats by the 4 vessel occlusion method for 10 min and was followed by different periods of reperfusion. Neuron loss was preceded by a rapid increase in the generation of ROS, NO, and lipid peroxidation, as well as by a decrease in Na+K+-ATPase activity and disrupted antioxidant defenses in the hippocampus and cortex. Pretreatment with resveratrol showed significantly attenuated neuronal death in the hippocampus and cortex and a decrease in generation of ROS, lipid peroxidation and NO content. Resveratrol also normalized Na K-ATPase activity in the cortex and hippocampus [32]. Moreover, resveratrol has been found to induce HO1 in a dose- and timedependent manner in cultured mouse cortical neuronal cells and to provide neuroprotection, which was lost when cells were treated with a HO inhibitor. Hence, resveratrol pretreatment protected mice subjected to an optimized ischemic-reperfusion stroke model, whereas mice in which HO1 was selectively deleted lost most of the beneficial effects [33]. Another study investigated the neuroprotective effect of upregulated Nrf2/ARE by resveratrol pretreatment in rats with focal cerebral ischemic injury. Ischemia was induced by middle cerebral artery occlusion (MCAO). Twenty four hours after reperfusion, the neurological score, the infarct volume, and the brain water content were measured. The result obtained was that resveratrol pretreatment had significantly upregulated the expression of Nrf2, HO-1 and SOD. As a consequence, ameliorated neurological scores, reduced infarct volume, brain water content, and decreased MDA levels were seen in rats pretreated with the compound. In addition, the expression of caspase-3 was downregulated along with decreased DNA fragmentation. This neuroprotective effect could be due to upregulated expression of Nrf2 and HO-1 [34]. A different study showed that resveratrol could offer protection against ischemic injury by improving brain energy metabolism and alleviating oxidative stress. Cerebral ischemia was induced by the use of the MCAO model. Then, it was found that ischemic infarcts were significantly reduced and neurological functions were improved in the resveratrol-treated group compared with the ischemia group. Results also revealed that resveratrol treatments had increased levels of glucose and ATP and lower levels of lactate during the ischemic period. The treatment also increased the basal levels of adesonine and inosine while inhibiting elevations of hypoxanthine and xanthine levels, as well as provoking a decrease in xanthine oxidase activity and MDA levels in the microdialysate of the hypothalamus [35]. The neuroprotective effect of resveratrol against mitochondrial dysfunctions in the hippocampus induced by brain injury has been investigated in the MCAO model. Resveratrol significantly restored ATP content and the activity of mitochondrial respiratory complexes while cytochrome c release was reduced in the resveratrol treated group. A marked decrease in DNA fragmentation was also perceived after resveratrol treatment. Histological analysis of CA1 hippocampal neurons revealed that resveratrol treatment diminished intercellular and pericellular edema and glial cell infiltration. Moreover, the brain infarct volume and brain edema were significantly reduced [36]. Extensive evidence suggests that blocking of the inflammatory reaction by resveratrol could be a possible mechanism of neuroprotection. The effect of resveratrol pretreatment on the NFκB inflammatory cascade and COX-2, iNOS and JNK levels in experimental ischemia has been investigated in rats subjected to 4vessel occlusion. Resveratrol pretreatment significantly reduced astroglial and microglial activation 7 days after ischemia, as well as greatly attenuating ischemia induced NF-κB and JNK activation


with decreased COX-2 and iNOS production. Results indicate that the suppression of the inflammatory response via regulation of NFκB, COX-2 and iNOS induced by injury could play a role in neuroprotection of resveratrol [37]. It was also demonstrated that resveratrol modulates cellular apoptosis in neurons. A study has actually investigated this effect in a rat brain ischemia model induced by MCAO. Results revealed that resveratrol treatment increased the expression of anti-apoptotic protein Bcl-2 and decreased the expression of pro-apoptotic protein Bax in the MCAO rats. Additionally, resveratrol treatment significantly reduced brain infarct volume along with DNA fragmentation of hippocampal CA1 neurons in the rats. The reason is that MCAO operation impaired long-term potentiation in the hippocampal CA1 region and the basal synaptic transmission between the Schaffer collaterals, which was also rescued by resveratrol treatment. The neuroprotective qualities of resveratrol may be attributable to the upregulation of Bcl-2 expression and downregulation of Bax expression [38]. Resveratrol is also a direct activator of sirtuin 1 (SIRT1), related to increased lifespan in various species similar to calorie restriction, thereby providing significant neuroprotective benefits. Recent studies in a variety of species, including mammals, showed that both resveratrol treatment and caloric restriction enhanced silent information regulator 2/sirtuin 1 activity, which mediated an increase in life span/cell survival. The protective effects of resveratrol are mediated by Akt and mitogen-activated protein kinases. The signaling pathway modulated by resveratrol in ischemia involving SIRT1 expression and phosphorylation of Akt, extracellular-signal-regulated kinases1/2 (ERK1/2), and p38 in the ischemic cortex has also been investigated. Resveratrol has been found to increase the expression of SIRT1 and phosphorylation of Akt and p38 while inhibiting the increase in phosphorylation of ERK1/2. An elevation in the levels of peroxisome proliferatoractivated receptor (PPAR) γ coactivator 1α and SOD2 was also detected [39]. Further investigation of the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways was carried out in an experimental model of global cerebral ischemia. During this procedure, ischemia was induced in rats by the 4-vessel occlusion method for 10 min, which was followed by different periods of reperfusion. Pretreatment with resveratrol significantly reduced neuronal death, while pretreatment with this compound increased the phosphorylation of Akt, GSK-3β and CREB in the CA1 hippocampus after ischemia. However, the administration of the PI3-K inhibitor, LY294002, compromised the neuroprotective effect of resveratrol and decreased the level of pAkt, p-GSK-3β and p-CREB after ischemic injury. This result indicates that resveratrol could protect against delayed neuronal death in the hippocampal CA1 neurons by maintaining the prosurvival states of Akt, GSK-3β and CREB pathways [40]. Akt is a serine/threonine-specific protein kinase that plays a key role in various cellular processes, such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration. Ischemic preconditioning (IPC) is a technique for producing resistance to the loss of blood supply. Resveratrol is currently the focus of intense research as an IPC agent in kidney, heart, and brain. Results indicate that pharmacological preconditioning with resveratrol was attributed to its role as an intracellular antioxidant and anti-inflammatory agent, as well as to its ability to induce NOS expression and angiogenesis, and to increase SIRT1 activity. PPAR γ co-activator-1alpha (PGC-1α) is a member of a family of

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transcription co-activators that presents activities on mitochondrial biogenesis, antioxidation, growth factor signaling regulation, andangiogenesis. This protein interacts with PPAR-γ, allowing the interaction of this protein with multiple transcription factors. It can interact with and regulate, for instance, the activities of CREB [41]. Many signaling pathways activated by resveratrol involve PGC-1α activity. Resveratrol can even exert pharmacological preconditioning by activating PGC-1α. Inhibition of SIRT1 has been found to abolish ischemic preconditioning-induced neuroprotection in the CA1 region of the hippocampus [42]. In an in vitro model of cerebral ischemia employing organotypic hippocampal slice culture, resveratrol pretreatment was found to mimic IPC via the SIRT1 pathway. Therefore, the blockade of SIRT1 activation by sirtinol after either IPC or resveratrol pretreatment jeopardized their neuroprotection [43]. Oral resveratrol treatment has been found to protect cerebral neurons from enhanced damage produced by recurrent stroke, which is mediated in part by a protective effect of resveratrol on the endothelium of the cerebrovasculature. Experiments with adult rats have demonstrated that brain damage is exacerbated when a mild ischemic stroke is followed by a second mild cerebral ischemia. It was also shown that daily oral resveratrol treatment after the first ischemic insult reduced the ischemic cell death in such cases. Further investigation demonstrated reduction of both inflammatory changes and markers of oxidative stress in resveratrol treated animals. Investigation of resveratrol effects on the BBB in vivo demonstrated that resveratrol treatment reduced BBB disruption and edema following recurrent stroke without affecting regional cerebral blood flow. Investigation in primary cell culture studies demonstrated that resveratrol treatment significantly protected endothelial cells against ischemia, resulting in improved viability against oxygen and glucose deprivation. SIRT-1 inhibition with sirtinol partially improved cell viability following oxygen glucose deprivation, suggesting that endothelial protection could be SIRT-1 dependent [44]. Mitochondrial uncoupling proteins (UCP) are members of the larger family of mitochondrial anion carrier proteins (MACP). UCPs separate oxidative phosphorylation from ATP synthesis with energy being dissipated as heat, also referred to as the mitochondrial proton leak. UCPs facilitate the transfer of anions from the inner to the outer mitochondrial membrane and the return transfer of protons from the outer to the inner mitochondrial membrane. They also reduce the mitochondrial membrane potential in mammalian cells. Evidence demonstrates that neuroprotection by resveratrol pretreatment involves alterations in mitochondrial function via the SIRT1-UCP2 pathway. Significantly decreased UCP2 levels were found in hippocampal mitochondria isolated 48 h after resveratrol treatment. However, the introduction of the SIRT1-specific inhibitor sirtinol abolished the neuroprotection afforded by resveratrol treatment and the decrease in UCP2 levels. Resveratrol treatment also significantly increased the ADP/O ratio in hippocampal mitochondria, reflecting enhanced ATP synthesis efficiency [45]. Resveratrol could exert a neuroprotective effect against ischemic injury by modulating the release of neurotransmitters and neuromodulators during ischemia. This was pointed out in a study carried out in MCAO ischemic rats, in which dialysates of hypothalamus were obtained by the brain microdialysis technique. Ischemic infarcts were significantly reduced and neurological functions were improved in the resveratrol-treated group compared with the ischemia group. The analysis results demonstrate that chronic treatment with resveratrol remarkably reduced the release of


the excitatory neurotransmitter glutamate, aspartate and the neuromodulator D-serine during ischemia and reperfusion. On the other hand, it significantly increased the basal extracellular levels of the inhibitory neurotransmitter -amino-n-butyric acid (GABA), glycine and taurine, as well as ameliorating the excitotoxic index during ischemia and reperfusion [46]. Ginkgo biloba: Ginkgo biloba extracts are now prescribed in several countries for their reported health benefits, particularly for medicinal properties in the brain. The standardized Ginkgo extract, EGb761, has been reported to protect neurons against oxidative stress. A study performed with rats evaluated the LPO and the activity of catalase and SOD in the hippocampus, striatum and substantia nigra treated with EGb761. The result obtained was an increase in catalase and SOD and a decrease of the LPO activity in the hippocampus, striatum and substantia nigra [47]. In a study using a model of transient global ischemia-induced delayed hippocampal neuronal death and inflammation, mice were subjected to an 8-min bilateral common carotid artery occlusion (tBCCAO). The experiment demonstrated that EGb761 pretreatment reduced DNA fragmentation of hippocampal neurons. The reason is that pretreatment with EGb761 upregulated the expression levels of HO1, Nrf2, and vascular endothelial growth factor (VEGF). Also, the number of activated astrocytes and microglia was found to be significantly lower in the EGb761 pretreated group. Results advocate that neuroprotection by EGb761 could be related to activation of the HO1/Nrf2 pathway, upregulation of VEGF and downregulation of inflammatory mediators such as astrocytes and microglia [48]. In addition, EGb761 has shown significant neuroprotection in the permanent distal middle cerebral artery occlusion (pMCAO) model. Mice subjected to pMCAO and treated 4 h later with 100 mg/kg EGb761 showed significantly lower infarct volumes and neurologic deficit score (NDS). HO1, VEGF and eNOS levels in the brain cortices were higher in EGb 761 treated mice, whereas HO1 knockout (HO1⁻/⁻) mice showed significantly higher infarct volume and NDS. HO1⁻/⁻ mice showed no neuroprotection when treated with EGb761 [49]. The neuritogenic potential of EGb761 has been investigated in mice pretreated with EGb761 for 7 days and then subjected to transient middle cerebral artery occlusion (tMCAO) and 48 h of reperfusion. Significant reductionwas observed in infarct volumes and neurologic deficits. Pretreatment with EGb761 significantly enhanced the survival of primary neurons exposed to tertiary butylhydroperoxide (t-BuOOH), H2O2, and NMDA. In addition, it significantly increased the protein expression levels of Nrf2, HO1, GAPDH, β-actin, CRMP2, and histone H3 during t-BuOOHinduced oxidative stress. Bilobalide and ginkgolide A were also found to protect cells against NMDA-induced excitotoxicity. These findings suggest that EGb761 has neuritogenic potential [50]. EGb761 has been found to prevent ischemia-induced impairment of Na, K-ATPase activity, which is implicated in the pathophysiology of cerebral ischemia. A study compared ATPase activity and expression between the ischemic ipsilateral cortex and the nonischemic contralateral cortices 1 h after unilateral occlusion of the middle cerebral artery in mice. The ipsilateral cortex had lower K-ATPase activity and higher lipid peroxidation when compared with the contralateral cortex. Nevertheless, pre-treatment with EGb761 extinguished the differences observed between ipsilateral and contralateral cortex [51].


EGb761 reduces the formation of cellular edema and neurodegeneration under conditions of ischemia. In a study investigating the effect of EGb761, oxygen-glucose deprivation in brain slices and MCAO were used to induce ischemia in mice brains. EGb761 reduced ischemia-induced cell swelling in hippocampal slices by 60% and cell degeneration and edema formation after MCAO by around 50%. Pretreatment with EGb761 prevented the increase in striatal glutamate levels. Reduction of excitotoxicity could be a possible mechanism of action for the neuroprotective action of EGb761 [52]. In order to investigate further the neuroprotective mechanisms of EGb761, its effects on NMDA excitotoxicity and focal cerebral ischemia were studied in cell suspensions of neonatal rat hippocampus. Twelve days after incubation, the suspension of neurons was divided into 4 groups: a normal control group, a NMDA group, a MK-801 (an NMDAR antagonist) group and an EGb761 pretreatment group. The cell viability of the EGb761 group was significantly higher than that of the NMDA group and significantly lower than that of the MK-801 group. In turn, lactate dehydrogenase efflux of the EGb761 group was significantly lower than that of the NMDA group and significantly higher than that of the MK-801 group. It was possible to perceive a significant inhibition of NMDA-activated inward current in the EGb761 group. Hence, results indicate that neuroprotection by EGb761 could be related to decreasing NMDA excitotoxicity [53]. Further studies to find the effect of EGb761 on rats during ischemia and its influence on intracellular calcium in hippocampal neurons indicate that it provokes a decrease in glutamate and aspartate and an increase in GABA concentration. Administration of EGb761 significantly inhibited the effect of glutamate on calcium currents. Thus, it could protect neurons by keeping the balance of inhibitory/excitatory amino acids and reducing excitotoxicity induced by calcium currents [54]. Ginkgolide B, a purified terpene lactone component extracted from Ginkgo biloba leaves, is used as an antioxidant and antagonist of platelet activating factor (PAF) receptors. Pretreatment with ginkgolide B in MCAO rats caused a reduction in the ischemia-induced elevation of levels of glutamate, aspartic acid and glycine, an increase in extracellular GABA, a decrease in the excitotoxic index and a reduction in the volume of cerebral infarction [55]. EGb761 has been found to enhance significantly the phosphorylations of Akt, CREB and the expression of brain-derived neurotrophic factor (BDNF) in rat brains [56]. A study used the MCAO model in rats to investigate whether the neuroprotective effects of EGb761 are modulated through Akt and its downstream target, Bcl-2-associated death promoter (BAD). When BAD is phosphorylated by Akt, it leaves Bcl-2 free to inhibit Bax-triggered apoptosis, having an anti-apoptotic effect. It was possible to notice that EGb761 significantly reduced infarct volume. Then, potential activation was measured by phosphorylation of Akt at Ser (473) and BAD at Ser (136). EGb761 prevented the injury-induced decrease of phosphorylated Akt and phosphorylated BAD. Furthermore, EGb761 prevented the injury-induced increase of cleaved caspase-3 levels [57]. The ability of EGb761 to regulate the expression of PEA-15 and two of its phosphorylated forms (Ser 104 and Ser 116) in MCAO induced injury has also been studied. PEA15 is a death effector domain (DED)-containing protein predominantly expressed in the CNS, particularly in astrocytes. PEA-15 is an endogenous substrate for protein kinase C that modulates cell proliferation and apoptosis. Phosphorylation of PEA-15 influences its anti-apoptotic function. As a consequence, a decrease in PEA-15 phosphorylation induces


apoptotic cell death. During the experiment, a reduction in expression of PEA-15 and its phosphorylated forms induced by MCAO injury was detected. However, the EGb 761 pretreatment prevented this ischemic injury-induced decrease. In addition, EGb761 attenuated the injury-induced reduction in phosphorylated PEA-15 (Ser 104) and phosphorylated PEA-15 (Ser 116). The maintenance of PEA-15 phosphorylation by EGb761 pretreatment during cerebral ischemic injury indicates its involvement in the neuroprotective mechanism of EGb761 [58]. EGb761 has also been seen to decrease significantly Bax/Bcl-2 ratios in all brain regions in young and senescence-accelerated mice following global ischemia, indicating its role in the modulation of apoptosis [59]. Ginkgolide K isolated from the leaves of Ginkgo biloba is a natural platelet-activating factor receptor antagonist and, therefore, its effect on neuroprotection in cerebral ischemia has been investigated in the MCAO model. It was noticed that pretreatment with ginkgolide K significantly diminished the volume of infarction and brain water content, and improved NDS, as well as markedly reversing the level of MDA, NO, NOS and SOD to their normal state in either serum or cerebral ischemic section. The neuronal injury was significantly improved after pretreatment with ginkgolide K [60]. Curcumin: Curcumin is the principal curcuminoid of the popular Indian spice turmeric, which is a member of the ginger family (Zingiberaceae). The curcuminoids are polyphenols responsible for the yellow color of turmeric. Chemically speaking, curcumin is a diferuloylmethane that has a diferulic acid moiety fused with another carbon atom or methylene moiety. Thus, it has a methylene1,3-diketo group showing keto-enoltautomerism due to stabilization by hydrogen bonding. Curcumin exists mainly in the keto-enol form rather than in a diketo form. Potential protective effects of curcumin against ischemic insult in rat forebrain have been investigated using the BCCAO model. Curcumin was found to decrease ischemia-induced elevation of xanthine oxidase (XO) activity, superoxide anion production, MDA level and the activities of glutathione peroxidase (GPx), SOD and LDH, thus protecting rat forebrain against ischemic injury [61]. Treatment of rats subjected to transient forebrain ischemia with curcumin caused a decrease in MDA levels and an increase in GSH contents, catalase and SOD activities, which returned to normal levels [62].

Ghosh et al.

of redox potential. Besides upregulating Nrf2 and HO-1 in MCAOaffected brain tissue, curcumin treatment has been found to reduce infarct volume, brain water content and behavioral deficits caused by MCAO. Taking these results into account, the protection of the brain from damage caused by MCAO may be attributed to upregulation of Nrf2 expression [66]. Curcumin administration significantly decreased ischemia-induced neuronal death, astrocytes and microglial activation, lipid peroxidation, mitochondrial dysfunction and apoptotic indices (increased cytochrome c release followed by caspase-3 activation) in hippocampal CA1 neurons of gerbils subjected to global cerebral ischemia by transient occlusion of the common carotid arteries [67]. The effects of curcumin in focal cerebral ischemia in rats also indicate its antiapoptotic activity. Curcumin treatment reduced cytochrome c and caspase 3 expressions and increased mitochondrial Bcl-2 expression [68]. In order to explore the anti-inflammatory effect of curcumin in cerebral ischemic injury, a study was conducted in gerbils by BCCAO induced forebrain ischemia. That work provided an evaluation of the effect of curcumin use on Jun and NF-κB expression. The number of apoptotic neurons in the hippocampal CA1 region was lowered in curcumin treated gerbils, as well as the expression of Jun and NF-κB in CA1 area. On the other hand, the expression of Fos increased in those gerbils. A further study aimed to investigate the effects of curcumin on the expression of NF-κB and intercellular adhesion molecular 1 (ICAM-1) was conducted in rats with cerebral ischemia-reperfusion injury. The contour of the pyramidal cells in the hippocampal CA1 region was distorted after the injury, which also led to increased levels of NF-κB and ICAM-1. Curcumin substantially ameliorated cerebral pathological changes as more than 70% of the pyramidal cells retained distinct cell contour and nuclear boundary in curcumin treatment rats. That group also had their levels of NF-κB and ICAM-1 reduced [69]. These results indicate that anti-inflammatory activity of curcumin contributes to its protective mechanisms. A study of curcumin administration in cultured astrocytes indicates its role in protecting the BBB integrity. The compound significantly inhibited iNOS expression in these cells, as well as preventing ONOO- induced damage to brain capillaries in ipsilateral hemisphere endothelial cells. Such results indicate that curcumin ameliorates cerebral ischemia injury by preventing ONOOmediated BBB damage [70].

Curcumin has exhibited neuroprotection in the case of MCAOinduced focal cerebral IR injury. The administration of curcumin after MCAO provoked a reduction in infarct volume and cerebral edema. The increase in lipid peroxidation in ipsilateral and contralateral hemispheres of the brain observed after MCAO was reduced by curcumin treatment. Another effect of this treatment was the decrease in SOD and GPx activities in the ipsilateral hemisphere. Curcumin treatment also reduced peroxynitrite formation and hence the extent of tyrosine nitration in the cytosolic proteins. These results suggest that curcumin has a neuroprotective potential mediated through its antioxidant activity [63]. In a similar study, significant inhibition in lipid peroxidation and intracellular calcium levels was observed following treatment with curcumin in MCAO rats, along with increase in SOD activity in the corpus striatum and cerebral cortex [64].

Epigallocatechin-3-gallate (EGCG): Fresh tea (Camellia sinensis) leaves contain a high amount of catechins, a group of flavonoids or flavanols known to constitute 30-45% of the solid green tea extract. The favorable properties ascribed to tea consumption are believed to rely on its bioactive components, namely catechins and their derivatives. These components have been demonstrated to act directly as radical scavengers and exert indirect antioxidant effects by activating transcription factors and antioxidant enzymes. The most abundant polyphenolic compound is EGCG. It contributes to the beneficial effects attributed to green tea, such as its anticancer, cardiovascular function improvement and antioxidant antiinflammatory properties. As a consequence, green tea polyphenols are now being considered as therapeutic agents in well controlled epidemiological studies aimed to alter the brain aging processes and to serve as possible neuroprotective agents in progressive neurodegenerative disorders.

Curcumin stimulates HO-1 activity, which requires the activation of the Nrf2/ARE pathway. In fact, it promotes inactivation of the Nrf2Keap1 complex, leading to a higher level of Nrf2 activity [65]. Nrf2, in turn, coordinates the expression of genes required for free radical scavenging, detoxification of xenobiotics, and maintenance

EGCG induces expression of GST, GPX, GCL, HO-1, which are involved in the elimination or inactivation of ROS by activation of Nrf2 [71]. A study investigated the cytoprotective effects of EGCG against oxidative stress damage in a cell line of rat neurons. Immortalized rat neurons (H19-7) were exposed to various


concentrations of EGCG. After treatments, cells were harvested in order to determine HO-1 and Nrf2 activity. An elevated expression of HO-1 was detected in cultured neurons and its induction relates to the activation of Nrf2. Pre-incubation with EGCG, in turn, resulted in an enhanced cellular resistance to glucose oxidasemediated oxidative damage. However, this effect was significantly attenuated by zinc protoporphyrin IX, an inhibitor of HO-1 activity. This demonstrates that EGCG induced HO-1 expression, which was able to protect against oxidative stress-induced cell death in cultured neurons [72]. A recent study in gerbils has demonstrated the neuroprotective effect of EGCG against neuronal damage following global ischemia induced by BCCAO. When administered at a dose of 25 or 50 mg/kg, EGCG significantly reduced hippocampal CA1 neuronal damage in a dose-dependent manner [73]. In a different model of ischemia induced by MCAO in rats, EGCG reduced focal ischemia/reperfusion-induced brain injury. Fifty mg/kg EGCG significantly reduced the infarction volume and NDS along with levels of MDA and oxidized/total glutathione ratio [74]. In addition, it has been found to inhibit increased NO concentrations in the intact rat hippocampus subjected to ischemia. Another study has shown that EGCG reduced the effects of the NO donor, sodium nitroprusside (SNP), which decreases the viability of cultured rat hippocampal neurons. This could be important as NO plays a significant role in disrupting integrity of the BBB in ischemic conditions [75]. It was possible to observe a significant increase in the expression of nicotinamide adenine dinucleotide phosphate diaphorase/neuronal nitric oxide synthase (NADPH-d/nNOS) in neurons after hypoxic conditions. However, the hypoxia-induced increase in NADPH-d/nNOS expression was significantly depressed in the hypoxic rats treated with EGCG, which suggests that EGCG may attenuate the oxidative stress following acute hypoxia [76]. EGCG also attenuated NADPH-d/nNOS expression in motor neurons of rats that suffered peripheral nerve injury [77]. The beneficial effects of EGCG on excitotoxic neuronal damage have been investigated. In an in vitro study, excitotoxicity was induced by incubating neurons with 10 μM of NMDA. EGCG increased neuronal viability and reduced NMDA induced MDA production. EGCG attenuated the increase in MDA level caused by unilateral cerebral ischemia, which had been induced by occlusion of the right common carotid artery in rats. EGCG also reduced the formation of postischemic brain edema and infarct volume [78]. The effects of EGCG on high frequency stimulation-induced longterm potentiation (LTP) in the Schaffer collateral-CA1 synapse have been investigated both with and without cerebral ischemia injury induced by MCAO. The goal was to examine the possible relations between EGCG and synaptic transmission. The result was that EGCG improved efficiency of synaptic transmission in cerebral ischemia injury and regulated excitatory and inhibitory amino acid balance. The application of EGCG modulated synaptic transmission produced a dose-dependent improvement of the induction of LTP. It also improved cell viability of primary cultured rat hippocampal and cortical neurons subjected to oxygen-glucose deprivation, as well as normalizing the levels of glutamate, glycine, and GABA after the distortion that followed cerebral ischemic injury [79]. Besides, EGCG has anti-inflammatory effects in BV-2 microglia cells, and was shown to inhibit lipopolysaccharide- (LPS-) induced NO production and reduce COX-2 and iNOS expression in those cells [80]. EGCG's neuroprotective effects in cerebral ischemia may involve a modulation of MMPs. In a study to evaluate the effect of EGCG on MMP, C57BL/6 mice were subjected to 20 min of transient global


cerebral ischemia followed by investigation of MMP expression using gelatin gel zymography. In situ zymography detected ischemic gelatinase activity in pyramidal neuronal areas after brain ischemia, indicating expression of MMP-9. It was possible to observe neuronal DNA damage in CA1 and CA2 pyramidal sectors. However, mice treated with EGCG showed significantly reduced gelatinase levels and neuronal damage. Hence, results demonstrate that EGCG suppresses MMP-9 activation [81]. Besides, there is evidence suggesting that EGCG could modulate apoptosis through the mitochondria-mediated apoptosis pathway involving the Bcl-2 family. A study was designed to investigate possible mechanisms of EGCG-mediated inhibition against the apoptosis caused by exposure to CoCl2 in rat pheochromocytoma PC12 cells. The exposure to CoCl2 caused generation of ROS, as well as inducing cell death with apoptotic morphology and DNA fragmentation. However, EGCG diminished the loss of viability in the cells exposed to CoCl2 and led to a reduction in DNA fragmentation. Also, EGCG attenuated the CoCl2 induced disruption of mitochondrial membrane potential and the release of cytochrome c from the mitochondria to cytosol, besides abolishing CoCl2-stimulated activities of caspase-9 and caspase-3. In addition, EGCG inhibited the decrease of the Bcl-2/Bax ratio induced by CoCl2 treatment [82] and has been found to attenuate the reduced activation and expression of ERK, p38 MAPK, and Akt induced by H2O2 in HLEB-3 cells, a human lens epithelial cell line. Other effects attributed to EGCG are the inhibition of the H2O2-stimulated increase of caspase-9 and caspase-3 expression and the decrease of the Bcl-2/Bax ratio [83]. EGCG also inhibits NO-induced apoptosis in rat PC12 cells. The administration of SNP decreased the cell viability and induced apoptosis, showing cell shrinkage and chromatin condensation as well as subG1 fraction of cell cycles. The compound has additionally been shown to inhibit cytotoxicity and apoptotic morphogenic changes induced by SNP, and to decrease the Bcl2/Bax ratio, as well as attenuating the production of ROS by SNP. EGCG prevented the release of cytochrome c from the mitochondria into the cytosol and inhibited activation of caspase-9 and caspase-3 induced by SNP [84]. Baicalin: Baicalin, a flavonoid isolated from the plant Scutellaria baicalensis, has a wide range of pharmacological activities. Besides being a potent anti-inflammatory and anti-tumor agent, it was shown to be effective against cerebral ischemia-reperfusion injury in a study involving rats with focal cerebral ischemia. Infarct volume was significantly reduced by baicalin treatment, which increased expression of BDNF and reduced expression of caspase-3 [85]. The neuroprotective effect of baicalin on permanent cerebral ischemia injury, in turn, has been determined in rats subjected to permanent middle cerebral artery occlusion (pMCAO). The treatment with this compound was found to reduce neurological deficit scores and cerebral infarct volume after pMCAO, as well as causing a significant decrease in the enzymaticactivity of myeloperoxidase and in the expression of iNOS and COX-2 mRNA in rat brain. Also, it significantly inhibited neuronal apoptosis and the expression of cleaved caspase-3 protein after pMCAO [86]. Cerebral ischemia-reperfusion can activate several transcriptionfactors and lead to inflammatory reactions related to pattern recognition receptors with immune activating functions. NOD2 (nucleotide-binding oligomerization domain protein 2) is one of the receptors involved in innate immune response and is genetically associated with several inflammatory reactions. In this context, a study was conducted to assess the effect of baicalin on NOD2 in cells subjected to oxygen-glucose deprivation in vitro and


Ghosh et al.

Table 1: Herbal lead molecules under investigation for treatment of cerebral ischemia. Molecules Ligustilide obtained from Angelica sinensis

Neuroprotective activity Ligustilide protects against permanent focal ischemic damage in rats by reducing the cerebral infarct volumes and significantly improving behavioral deficits [94]. Pretreatment of PC12 cells with Z-ligustilide attenuated H2O2 induced cell death, attenuated increased intracellular ROS levels, and decreased Bax expression, cleaved-caspase 3 and cytochrome-c [95].

Tanshinone obtained from Salvia miltiorrhiza

Tanshinone significantly increased the activity of SOD after 24 h of ischemia and decreased the MDA level, NO content, and iNOS expression [96]. Tanshinone IIB significantly reduced the focal infarct volume, cerebral histological damage and apoptosis in rats subjected to MCAO [97]. After tanshinone treatment of rats subjected to MCAO, the levels of glutamate were significantly lower in the hippocampus, and NMDAR1 optical density was reduced in the CA1 region [98].

Wogonin obtained Wogonin has been shown to decrease inflammatory activation of cultured brain microglia by diminishing LPS-induced TNF-α, IL-1β, and NO from Scutellaria baicalensis production.Wogonin attenuated the death of hippocampal neurons by transient global ischemia by four-vessel occlusion [99]. Wogonin inhibits microglial cell migrationto the ischemic lesion via suppression of NFκB activity [100]. Wogonin had a protective effect on neuronal cells damaged by oxygen and glucose deprivation in rat hippocampal slices in culture [101]. Paeonol obtained from Paeonia suffruticosa

Paeonol reduced cerebral infarct and neurodeficit in rats subjected to BCCAO. Paeonol suppresses and scavenges superoxide anion, and inhibits microglia activation and IL-1β in ischemia-reperfusion injured rats [102]. Paeonol protected rat hippocampal neurons from oxygen-glucose deprivation-induced injury by reducing the morphological damage and increasing neuron viability. Paeonol reversed the overload of NMDA induced intracellular Ca2+ [103].Reduced levels of Bax protein in mitochondria and of apoptosis-inducing factor (AIF) in cytosol were seen after paeonol treatment in rats subjected to transient MCAO [104].

Shikonin obtained from Treatment with shikonin reduced NDS, infarct size, levels of MDA and ROS and attenuated neuronal damage, as well as upregulating SOD, catalase, Lithospermum erythrorhizon GSH-Px activities [105]. Shikonins attenuate microglial inflammatory responses by inhibition of ERK, Akt, and NF-κB [106]. Scutellarin obtained from Erigeron breviscapus

Scutellarin significantly reduced infarct volume, ameliorated the NDS and reduced the permeability of the BBB. Up regulation of eNOS and downregulation of VEGF, bFGF and iNOS expression was observed with scutellarin treatment [107]. Scutellarin significantly increased SOD, catalase activities and GSH levels in ischemic brain tissues [108]. Scutellarin treatment reversed brain NAD depletion and reduced DNA fragmentation along with inhibition of PARP overactivation and AIF translocation from the mitochondria to the nucleus after cerebral ischemia in rats subjected to MCAO [109].

in mice that had suffered cerebral ischemia-reperfusion. With regard to oxygen-glucose deprived cells, including BV2, PC12 and primary neuron cells, baicalin treatment was observed to down regulate the expression of NOD2 and TNFα, normalizing their levels after up regulation caused by oxygen-glucose deprivation. These data demonstrated that the targeting of NOD2, especially in neurons directly might be attributed to the neural-protective effect of baicalin on cerebral ischemia reperfusion injury [87]. Baicalin has been found to inhibit the TLR2/4 signaling pathway in rat brain after permanent cerebral ischemia induced by MCAO. Along with reducing cerebral infarct area and infarct volume, baicalin reduced the expression of TLR2/4, NFκB, iNOS and COX2 in rat brain. Baicalin also attenuated the serum content of TNF-α and IL-1β [88]. In addition, investigations have revealed that levels of NF-κB p65 in the cortex were increased after ischemia reperfusion injury. Nonetheless, this elevated level of NF-κB p65 decreased significantly after baicalin treatment. This inhibition may be related to the neuroprotective effects of the compound [89]. The neuroprotective effects of baicalin in gerbils subjected to transient global cerebral ischemic-reperfusion injury have also been studied. Baicalin administration significantly attenuated ischemia-induced neuronal cell damage and reduced level of MDA, and elevated activities of SOD, GSH and GSH-PX were found in baicalin-treated groups. Moreover, treatment with baicalin promoted the expression of BDNF and inhibited the expression of caspase-3. These findings suggest that baicalin's neuroprotection might be associated with its anti-oxidative and anti-apoptotic properties in global cerebral ischemia in gerbils [90]. Furthermore, the effect of baicalin on MMP-9 expression and BBB permeability following focal cerebral ischemia in rats with MCAO has been assessed. Neuronal damage, brain edema and BBB permeability were significantly reduced by baicalin administration after focal cerebral ischemia. Elevated expression of MMP-9 was significantly downregulated by baicalin administration, while the

decreased expression of occludin (tight junction protein) due to MCAO was significantly upregulated by baicalin administration [91]. In addition, the compound has been found to reduce the permeability of BBB during hypoxia in brain microvascular endothelial cells (BMVECs) from Bal b/c mice. The reason is that baicalin increases the expression of tight junction proteins in brain microvascular endothelial cells. Ischemia was stimulated by OGD, which led to a significant increase of permeability in this in vitro BBB model. The effects promoted by baicalin were: effective decrease in the permeability of the BBB, transcription and expression of claudin-5 and zonula occludens-1 (tight junction proteins), and reduction of the levels of PKC [92]. Conclusion: The development of protective agents from traditional herbal medicine is a promising direction in the treatment of ischemic cerebral injury and related neurodegenerative diseases. A better understanding of the cellular effects exerted by phytochemicals is vital to orientate a proper use of such compounds, which might be promising therapeutic agents. In this sense, future efforts will have to implement extensive methodological improvements to separate the real therapeutic value of these agents from unsubstantiated hopes associated with them. The active molecules must be isolated and tested through well-designed experiments followed by randomized, placebo-controlled studies to enable rational clinical use [93]. In Table 1, several molecular leads that may become the drugs of tomorrow are discussed so that future analysis can focus on natural compounds that have clear pharmacological targets and fewer side effects. Then, these phytochemicals may be used as lead molecules to synthesize synthetic derivatives with improved activity in order to develop new drugs against ischemia induced cerebral injury. Acknowledgement - The authors are thankful to the University Grants Commission, New Delhi for providing financial assistance to Dr. Subhash C. Mandal as UGC Research Awards (File no: F.301/2013(SA-II)/RA-2012-14-NEW-SC-WES-3684).

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Natural Product Communications Vol. 9 (7) 2014 Published online (www.naturalproduct.us)

A New Cytosporone Derivative from the Endophytic Fungus Cytospora sp. Tomoya Takano, Takuya Koseki, Hiromasa Koyama, and Yoshihito Shiono Synthesis and Biological Evaluation of Oseltamivir Analogues from Shikimic Acid Van Hung Nguyen, Van Cuong Pham, Thi Thao Do, Huong Doan Thi Mai, Nguyen Thanh Le, Van Nam Vu, Van Hieu Tran, Thi Minh Hang Nguyen, Wim Dehaen and Van Minh Chau Quantification and Comparison of Extraction Methods for Alkaloids in Aegle marmelos Leaves by HPLC Aniket Karmase, Prasanna K, Sruti Rasabattula and Kamlesh K Bhutani Stability of Capsaicinoid Content at Raised Temperatures Wenhui Si, Sun Wa Man, Zhen-Yu Chen and Hau Yin Chung PPZPMs - a Novel Group of Cyclic Lipodepsipeptides Produced by the Phytophthora alni Associated Strain Pseudomonas sp. JX090307 - the Missing Link between the Viscosin and Amphisin Group Hardy Weißhoff, Sarah Hentschel, Irmtraut Zaspel, René Jarling, Eberhard Krause and Thi Lam Huong Pham Chemical Composition of Vernonia albicans Essential Oil from India Rajesh K. Joshi Identification of Volatiles in Leaves of Alpinia zerumbet ‘Variegata’ Using Headspace Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry Jian Yan Chen, Zheng Mei Ye, Tian Yi Huang, Xiao Dan Chen, Yong Yu Li and Shao Hua Wu Chemical Composition and Biological Activities of the Essential Oil from Anredera cordifolia Grown in Brazil Lucéia Fátima Souza, Ingrid Bergman Inchausti de Barros, Emilia Mancini, Laura De Martino, Elia Scandolera and Vincenzo De Feo Profile of Volatile Components of Hydrodistilled and Extracted Leaves of Jacaranda acutifolia and their Antimicrobial Activity Against Foodborne Pathogens Abdel Nasser B. Singab, Nada M. Mostafa, Omayma A. Eldahshan, Mohamed L. Ashour and Michael Wink Chemical Composition, Antioxidant, Antimicrobial and Anti-inflammatory Activities of the Stem and Leaf Essential Oils from Piper flaviflorum from Xishuangbanna, SW China Ren Li, Jing-jing Yang, Yuan-fei Wang, Qian Sun and Hua-bin Hu Essential Oil Composition and Antifungal Activity of Aerial Parts of Ballota nigra ssp foetida Collected at Flowering and Fruiting Times Daniele Fraternale and Donata Ricci Essential Oils from Schinus Species of Northwest Argentina: Composition and Antifungal Activity Diego A. Sampietro, María Melina E. Belizan, Zareath P. Terán Baptista, Marta A. Vattuone and Cesar A. N. Catalán Anxiolytic-like Effect of Inhalation of Essential Oil from Lavandula officinalis: Investigation of Changes in 5-HT Turnover and Involvement of Olfactory Stimulation Mizuho Takahashi, Ayako Yamanaka, Chihiro Asanuma, Hiroko Asano, Tadaaki Satou and Kazuo Koike

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Accounts/Reviews Biological Properties of 6-Gingerol: A Brief Review Shaopeng Wang, Caihua Zhang, Guang Yang and Yanzong Yang Antioxidant Activity of Natural Products Isolated from Red Seaweeds Caio Cesar Richter Nogueira, Izabel Christina Nunes de Palmer Paixão and Valéria Laneuville Teixeira Bioactive Secondary Metabolites from Acid Mine Waste Extremophiles Andrea A. Stierle and Donald B. Stierle Advances in Herbal Medicine for Treatment of Ischemic Brain Injury Nilanjan Ghosh, Rituparna Ghosh, Zulfiqar A Bhat, Vivekananda Mandal, Sitesh C. Bachar, Namsa D. Nima, Otimenyin O. Sunday and Subhash C. Mandal

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Natural Product Communications 2014 Volume 9, Number 7 Contents Original Paper

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Three New Monoterpene Glucosides from Senecio solidagineus Dingxiang Li, Guixin Chou and Zhengtao Wang Two Novel Iridoids from Morinda longifolia Ninh Khac Ban, Vu Huong Giang, Tran My Linh, Le Quynh Lien, Ninh Thi Ngoc, Le Duc Dat, Nguyen Phuong Thao, Nguyen Xuan Nhiem, Nguyen Xuan Cuong, Van Cuong Pham, Nguyen Hoai Nam, Jacinto Regalado, Huynh Van Keo, Phan Van Kiem and Chau Van Minh Antifeedant and Phagostimulant Activity of Extracts and Pure Compounds from Hymenoxys robusta on Spodoptera exigua (Lepidoptera: Noctuidae) Larvae Zaida N. Juárez, Antonio M. Fortuna, Eugenio Sánchez-Arreola, Jesús F. López-Olguín, Horacio Bach and Luis R. Hernández Chemical Evidence for the Liverwort Complex, Chiloscyphus concavus and C. horizontalis Jorge Cuvertino-Santoni, Yoshinori Asakawa, Denilson F. Peralta and Gloria Montenegro Chemical Constituents of Marrubium vulgare as Potential Inhibitors of Nitric Oxide and Respiratory Burst Farzana Shaheen, Shagufta Rasool, Zafar Ali Shah, Samreen Soomro, Almas Jabeen, M. Ahmed Mesaik and M. Iqbal Choudhary Abietane Diterpenoids from Clerodendrum trichotomum and Correction of NMR Data of Villosin C and B Linzhen Li, Long Wu, Menghua Wang, Jianbo Sun and Jingyu Liang Theoretical Research into Anticancer Activity of Diterpenes Isolated from the Paraiban Flora Luciana Scotti, Hamilton Ishiki, Francisco J.B. Mendonça Junior, Paula F.Santos, Josean F. Tavares, Marcelo S. Silva and Marcus T. Scotti Seed Dormancy Breaking Diterpenoids from the Liverwort Plagiochila sciophila and their Differentiation Inducing Activity in Human Promyelocytic Leukemia HL-60 Cells Hiromichi Kenmoku, Hiroyuki Tada, Megumi Oogushi, Tomoyuki Esumi, Hironobu Takahashi, Masaaki Noji, Takeshi Sassa, Masao Toyota and Yoshinori Asakawa Application of Microalgal Fucoxanthin for the Reduction of Colon Cancer Risk: Inhibitory Activity of Fucoxanthin Against β-Glucuronidase and DLD-1 Cancer Cells Arthitaya Kawee-ai and Sang Moo Kim Synthetic and Structure-Activity Relationship of Insecticidal Bufadienolides Ace Tatang Hidayat, Achmad Zainuddin, Danar Dono, Wawan Hermawan, Hideo Hayashi and Unang Supratman Cytotoxic Alkaloids from Leaves and Twigs of Dasymaschalon sootepense Sakchai Hongthong, Chutima Kuhakarn, Vichai Reutrakul, Surawat Jariyawat, Pawinee Piyachaturawat, Narong Nuntasaen and Thaworn Jaipetch The Effect of Zuccagnia punctata, an Argentine Medicinal Plant, on Virulence Factors from Candida Species Nuño Gabriela, Alberto María Rosa, Zampini Iris Catiana, Cuello Soledad, Ordoñez Roxana Mabel, Sayago Jorge Esteban, Baroni Veronica, Wunderlin Daniel and Isla María Ines Rare Prenylated Isoflavones from Tephrosia calophylla Seru Ganapaty, Vimal Nair, Devarakonda Rama Devi, Steve Thomas Pannakal, Hartmut Laatsch and Birger Dittrich Antioxidant Properties of Phenolic Compounds from Baccharis articulata and B. usterii Simone Quintana de Oliveira, Virgínia Demarchi Kappel, Viviane Silva Pires, Claiton Leoneti Lencina, Pascal Sonnet, José Cláudio F. Moreira and Grace Gosmann Effect of Silitidil, a Standardized Extract of Milk Thistle, on the Serum Prolactin Levels in Female Rats Raffaele Capasso Standardization of Solvent Extracts from Onopordum acanthium Fruits by GC-MS, HPLC-UV/DAD, HPLC-TQMS and 1 H-NMR and Evaluation of their Inhibitory Effects on the Expression of IL-8 and E-selectin in Immortalized Endothelial Cells (HUVECtert) Armond Daci, Markus Gold-Binder, Davide Garzon, Alessio Patea and Giangiacomo Beretta A New Antimicrobial Anthrone from the Leaf Latex of Aloe trichosantha Anwar Oumer, Daniel Bisrat, Avijit Mazumder and Kaleab Asres Preparative Production of Spinochrome E, a Pigment of Different Sea Urchin Species Olga P. Shestak, Victor Ph. Anufriev and Vyacheslav L. Novikov Phenylpropanoids and Furanocoumarins as Antibacterial and Antimalarial Constituents of the Bhutanese Medicinal Plant Pleurospermum amabile Phurpa Wangchuk, Stephen G. Pyne, Paul A. Keller, Malai Taweechotipatr and Sumalee Kamchonwongpaisan Spirocyclic Acylphloroglucinol Derivatives from Hypericum pyramidatum Rebecca Force, Shui Ling Chen, Emily Fortier, Emily Rowlands, Jean Heneks, David Rovnyak and Geneive E. Henry Anti-inflammatory Activity of Constituents Isolated from Terminalia chebula Min Hye Yang, Zulfiqar Ali, Ikhlas A. Khan and Shabana I. Khan Highly Potent Oligostilbene sbLOX-1 Inhibitor from Gnetum macrostachyum Serm Surapinit, Piyawit Sri-in and Santi Tip-pyang (Continued inside back cover)

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