Flavones from Root of Scutellaria Baicalensis Georgi - Ingenta Connect

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CNS & Neurological Disorders - Drug Targets, 2011, 10, 184-191

Flavones from Root of Scutellaria Baicalensis Georgi: Drugs of the Future in Neurodegeneration? Kazimierz Gsiorowski1, Eliza Lamer-Zarawska2, Jerzy Leszek3, Kalpana Parvathaneni4, Bharat Bhushan Yendluri4, Zofia Bach-Olszewska5 and Gjumrakch Aliev*,6 1

Department of Basic Medical Sciences, Faculty of Pharmacy, Wroclaw Medical University, 14 Kochanowskiego Str., 51-601Wroclaw, Poland 2

Department of Biology and Pharmaceutical Botany, Wroclaw Medical University, 14 Kochanowskiego Str., 51-601 Wroclaw, Poland 3

Department of Psychiatry, Wroclaw Medical University, 25 Kraszewskiego Str., 50–229 Wroclaw, Poland

4

Department of Biology, College of Sciences, The University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249 USA 5

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 25 Kraszewskiego Str., 50–229 Wroclaw, Poland 6

School of Health Science and Healthcare Administration, University of Atlanta, 6685 Peachtree Industrial Blvd, Atlanta, GA 30360, USA Abstract: Flavonoids are natural, plant-derived compounds which exert diverse biological activities, also valuable neuroprotective actions within the brain and currently are intensively studied as agents able to modulate neuronal function and to prevent age-related neurodegeneration. Among them, flavones isolated from Scutellaria baicalensis root exhibit strong neuroprotective effects on the brain and are not toxic in the broad range of tested doses. Their neuroprotective potential has been shown in both oxidative stress-induced and amyloid- and -synuclein –induced neuronal death models. Baicalein, the main flavone present in Scutellaria baicalensis root, strongly inhibited aggregation of neuronal amyloidogenic proteins in vitro and induces dissolution of amyloid deposits. It exerts strong antioxidative and anti-inflammatory activities and also exhibits anti-convulsive, anxiolytic, and mild sedative actions. Importantly, baicalein, and also another flavone: oroxylin A, markedly enhanced cognitive and mnestic functions in animal models of aging brains and neurodegeneration. In the preliminary study, wogonin, another flavone from Scutellaria baicalensis root, has been shown to stimulate brain tissue regeneration, inducing differentiation of neuronal precursor cells. This concise review provides the main examples of neuroprotective activities of the flavones and reveals their potential in prevention and therapy of neurodegenerative diseases.

Keywords: Neurodegeneration, baicalein, oroxylin A, wogonin, neuroprotection. INTRODUCTION Flavonoids are the large family of natural polyphenolic compounds abundant in the plant kingdom. More than 4000 flavonoids have been identified and isolated, and, probably, further compounds will be identified in the near future. The diversity of chemical composition and of spatial structure of flavonoids determine variability of their biological actions, however some general directions of their activity could be assembled. On the other hand, each flavonoid compound exerts multiple biological effects, since the compounds from this family are able to interfere with several intracellular signaling pathways which lead to a variety of biological effects [1, 2]. Many flavonoids exert a neuroprotective action by following four main molecular mechanisms: 1. Modulation of protein kinases and lipid kinase signaling pathways [2, 3] and suppression of an activation of pro-caspase-3 caused by oxidative stress [4] and as a result an inhibition of apoptosis and promotion of neuronal survival; 2. Decrease production of proinflammatory cytokines TNF- (Tumor Necrosis Factor-), IL-1, IL-6 (Interleukin-1 and 6) in activated microglia cells [5] and also lowering free radicals generation by direct inhibitory influence on inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and *Address correspondence to this author at the School of Health Science and Healthcare Administration, University of Atlanta, 6685 Peachtree Industrial Blvd, Atlanta, GA 30360, USA; Tel: 440-263-7461; E-mail: [email protected], [email protected]

1871-5273/11 $58.00+.00

NADPH oxidase in inflammatory reaction in astrocytes and microglia [6]; 3. Improvement of blood flow in the brain and induction of angiogenesis and, indirectly, stimulation of neurogenesis and neuron differentiation; and 4. An interaction with the molecular architecture of neurons responsible for memory and learning, including the structures involved in long-term potentiation and synaptic plasticity [3, 7]. Undoubtedly, flavonoids are valuable neuroprotective medicines and are worth further studying as potent drugs to be used in complementary therapies of neurodegeneration as well as precursor molecules to develop a new generation of drugs able to improve the brain functions [1]. FLAVONES OF SCUTELLARIA BAICALENSIS ROOT During the last decades special attention has been considered to flavonoid compounds isolated from traditional Asian medicines. Among them the dry root of Scutellaria baicalensis and the flavonoids isolated from the root have been intensively studied, since they exhibit a broad range of therapeutic benefit and no signs of toxicity even in high clinical doses. Among several classes of flavonoids are flavones which are present in roots of Scutellaria baicalensis. Flavones have been evaluated mainly for antioxidant, anti-inflammatory activity and also for their valuable neuroprotective effect. Scutellaria baicalensis Georgi (chin.: Huang – Qin, ang.: Scullcap or Chinese golden root) is the perennial herb of the family © 2011 Bentham Science Publishers

Flavones from Root of Scutellaria Baicalensis Georgi

CNS & Neurological Disorders - Drug Targets, 2011, Vol. 10, No. 2

Labiatae, which is widely distributed in Eastern Asia. The dry root of Scutellaria baicalensis Georgi (Scullcap) is one of the most popular and multi-purpose herb used in traditional medicine in China, Japan and in other oriental countries. The observed biological activity of Scullcap is related mainly to flavonoid content in the root. More than 30 flavonoid compounds have been identified in the root, the main are flavones: baicalein and wogonin and their glucuronides: baicalin and wogonoside [8]. Previously, the plant material usually originated in Asia, however, Scullcap can be easily cultivated under European climate conditions and the plant harvested in Central Europe contained the same flavonoids and in similar yields as plants grown in Asia [8]. The main flavones isolated from Scullcap root growing in Central Europe were: baicalin (baicalein-7-0-glucuronide; 10% up to 14% of dry root mass), baicalein (5, 6, 7–trihydroxyflavon; up to 5%), wogonin (5, 7-dihydroxy-8-methoxyflavon; 0,7%), wogonoside (wogonin-7-0glucuronide; 4,0%), oroxylin A (5, 7-dihydroxy-6-methoxyflavone), scullcupflavones I and II [8]. General chemical formula of the Scullcap flavones is given in Fig. (1).

2) [13]. There are two sites of the flavonoid absorption: the stomach/upper gastrointestinal tract –baicalin (native, glucuronic form) and the colon intestine – baicalein (aglycon, a product of degradation of baicalin by bacterial microflora enzymes) [13]. Both unconjugated baicalein and a small quantity of native baicalin absorbed by gut mucosa cells are extensively metabolized in liver and excreted in bile, whereas only a small portion of circulating metabolites are excreted with urine. The main metabolites present in plasma and the body fluids are glucuronides and sulfates [13].

R3 R2

B O

O A

C

OH

O

R1

Flavons:

R1

R2

R3

baicalein

OH

H

H

baicalin

OH

Glu

H

oroxylin A

OCH3

H

H

oroxylin A-glucuronide

OCH3

Glu

H

wogonin

H

H

OCH3

wogonoside

H

Glu

OCH3

Fig. (1). General chemical formula of the Scullcap flavones.

In herbal medicine special attention should be devoted for the standardization procedures of each Scullcap extract, as significant differences of flavone content between available products are noticed. For instance, baicalein concentration ranging from 0 to 52μg/mg of extracts commercially available in reputable Taiwan’s and Chinese companies was noticed [9]. As a criterion of standardization the content of baicalein in the extract is usually determined, and it is assumed that Scullcap raw material for herbal medicine purpose should contain no less than 8% of baicalein in dry root mass [10]. PHARMACOKINETICS OF THE SCULLCAP FLAVONES After oral administration of the aqueous extract of Scullcap, baicalein and wogonin are rapidly absorbed, whereas baicalin is metabolized into baicalein by intestinal bacterial microflora prior to intestinal absorption, and baicalein is detected in plasma up to 24 h [11, 12]. It was established that baicalin is poorly absorbed from the gastrointestinal tract in its native, glucuronic form, and must be hydrolyzed by intestinal bacterial beta-glucuronidase to its aglycone, baicalein, as was established in humans and rats [13]. Baicalein is readily absorbed into the intestinal mucosa cells and inside mucosa cells a major portion of baicalein is conjugated to baicalin by UDP-glucuronosyltransferase and then, the conjugate is transported into the blood, whereas more than half of the conjugate is excreted back to the intestinal lumen, mainly by mucosa cell membrane transporter-protein MRP2 (multi-drug resistance protein

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Baicalin enterohepatic recirculation comprises an intestinal conversion (bacterial enzyme-digestion) to baicalein, rapidly absorbed from the colon and transported to liver where it is converted back to baicalin by Phase 2 enzymes, and baicalin (being a secondary glucuronide) is secreted to bile, again enters intestinal tract. Thus the sustained level of baicalin in the systemic circulation is a results of absorption dynamics, and recirculation [14]. After intravenous administration of baicalein, about 75% of the dose was converted into the conjugated metabolites and the both glucuronides and sulfate conjugates of baicalein are present in blood plasma [14]. In rats the bioavailability of baicalin (native) was estimated to be 2.2% and of baicalin-conjugates was 27.8% [15]. The in vitro binding of baicalin to human plasma protein was estimated to be 86%-92% [16]. Baicalin is readily distributed to many tissues and organs in the human body [11]. Experiments in rats proved that baicalein (aglycone) entered brain crossing blood-brain barrier and was distributed in cerebral cortex, hippocampus, striatum, thalamus, cerebellum and brain stem within 20 min. after intravenous administration [11, 12]. Similar pharmacokinetics was established to wogonin and wogonoside [17], and it was pointed out that baicalin, wogonoside and other glucuronides of the Scullcap flavones were responsible for the biological effects of Scullcap root observed in vivo [13] Importantly, it was established that baicalin exhibited a strong impact on liver microsomal cytochrome P450 (CYP) monooxygenases; both an increase of CYP2B6 (in humans) and CYP1A1, 2B1 and 2C11 (in mice) [18], and a decrease of CYP2E1 (in mice) [19] were observed. Therefore, co-administration of baicalin with other medicines known to be substrates for cytochrome monooxygenases should be carefully monitored. PHARMACODYNAMICS OF CRUDE EXTRACT SCULLCAP ROOT, AND ISOLATED FLAVONES

OF

In traditional Chinese medicine crude extract from Scullcap root have been widely used for treatment of hyperlipidaemia, atherosclerosis, hypertension, common cold and inflammatory diseases such as atopic dermatitis, viral hepatitis, colitis and pancreatitis. The extract containing Scullcap flavones exerts direct anti-bacterial effect against a wide range of bacteria [20]. Scullcap has also been recognized as a mild relaxant that affects the neuronal and muscular-skeletal systems. Recently, several authors have shown that the Scullcap extract and its flavones exert a significant cytostatic effect on several human cancer cell lines in vitro and also in vivo in animal models [21-23]. Results of many investigations clearly indicated strong antioxidant effects of the Scullcap flavones on age-related redox imbalance, and documented their potent activities in prevention to the diseases connected with aging. Therefore, baicalin, baicalein, oroxyline A and wogonine have been widely used in therapy of oxidative stress in aging and immunological disorders, also they are considered as promising medicines in the treatment of acute cerebral ischemia and in prevention and alleviation of symptoms in neurodegenerative diseases, especially in Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). In fact, Scullcap root, crude extract and isolated flavones, especially baicalin and wogonoside, are considered an emerging multi-purpose, multi-therapeutic agent in modern medicine and pharmacy of Western countries [14].

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Although the precise mechanisms (probably diverse) and sites of intracellular action of compounds from the flavonoid family are largely unknown, it is suggested that their activity depends mainly on ability to: 1) Bind to extracellular receptors ERK1/2 (extracellular signal-regulated kinases), 2) Modulate the activity of MAP kinases (mitogen-activated protein kinases) and of PI3K lipid kinase (phosphatidylinositol-3 kinase), 3) Preserve Ca2+ homeostasis, preventing calcium-dependent activation of kinases in neurons and 4) Direct action on transcription factors and binding to promoter sequences [24]. More information is needed on the precise cellular sites of the Scullcap flavonoids action. For instance, it is still unclear, if their action requires cellular uptake or whether they are able of exert their effects by binding to extracellular receptor binding sites. It is a fundamental issue, since the metabolites, mainly secondary glucuronides predominate in body fluids, aglycone forms are present only in a very low content, and both baicalin, wogonoside and secondary glucuronides of baicalein and wogonin poorly penetrate across cellular membranes [24]. Therefore, their active transports into a cell and outside a cell are strongly suggested and several types of cellular transporter proteins are probably involved in their transport across cell membranes [2527]. It should be also stressed that flavonoid glucuronides posses different physico-chemical properties from their aglycones, and probably they also vary in their fate inside a cell. Learning on the Scullcap flavone pharmacodynamics in humans should be focused on analysis of baicalin and wogonoside effects on cells, as conjugated glucuronide action on cells probably predominate in vivo.

There is an ample evidence that flavonoids are effective in inhibition of oxidative stress induced neuronal injury, although their effect depends mainly on inhibition of free radical generating enzymes rather than on direct scavenging of radicals [1]. However, in the case of the Scullcap extract [34] and its four major flavones, a strong anti-oxidative effect and also a free radical scavenging activity were described in model in vitro experiments [35-40].

Although it is evident that the Scullcap flavones are potent, multi-therapeutic agents, a clear understanding of their intracellular fate and mechanism of action on cellular signaling pathways are important for future rational use in various therapies, including application to prevent and to treat neurodegeneration. Their antioxidative potential and anti-inflammatory activity are probably important mechanisms responsible for beneficial effects in neurodegeneration. ANTI-OXIDATIVE FLAVONES

ACTIVITIES

OF

THE

SCULLCAP

Oxidative stress is a well-recognized early response in chronic neurodegenerative diseases, especially in AD and in PD [25, 2832] Increased oxidative damage to all classes of biomolecules (lipids, proteins, sugars and nucleic acids) is regular future of ageing brain and is considered an important risk factor for neurodegeneration [28, 29]. Mitochondrial dysfunction, activation of microglia cells, environmental factors (ionizing radiations, metals, air pollutants, smoke and low antioxidant content in a diet etc.) generate oxidative stress in exposed cells, cause accumulation of free radical damage to cellular biomolecules which finally lead to cell death [30, 31]. Brain is prone to oxidative stress [28, 33], since the brain tissue contains high level of fatty acids (unsaturated fatty acids are susceptible to peroxidation) and also uses up to 20% of total body oxygen consumption, being relatively small by weight (2%) [33]. Also, human brain tissue has higher level of iron in certain regions (substantia nigra and basal ganglia) and aged brain contains increased level of redox metals (copper, iron, zinc) primed to catalyze the Fenton reaction of free radical cascade. On the other hand, brain is not adequately equipped with intrinsic antioxidant defense system, its antioxidant activity is markedly lower in comparison with other tissues. Thus the easiness of peroxidation of brain cell membrane lipids is a well documented, common attribute of neurons [33]. Currently, an efficient therapeutic strategy to preclude and to treat an oxidative damage is extensively searched for prevention of neurodegeneration and of many ageing-related diseases.

In the model system of in vitro generation of superoxide, hydroxyl and alkyl radical generation it was established by means of the electron paramagnetic resonance (EPR) analysis that baicalein was the most effective scavenger of radicals among the four tested Scullcap flavones. Baicalein and baicalin scavenged radicals in a dose-dependent manner, while wogonin and wogonoside did not exhibit this activity [35]. It was suggested that the presence of ortho-di-hydroxyl group in ring A of baicalein and baicalin determines their radical scavenging activity [35-36]. Another important mechanism of baicalein anti-free-radical effect is potency to chelate iron and the effectiveness in inhibition of Fenton reaction in vitro, in chemical model system which resemble the main physiological conditions [38]. In the same experiment baicalin (glucuronide) was not effective in iron chelation. The authors suggested that baicalein’s modulation of metal homeostasis and the inhibition of Fenton reaction was the possible mechanism of anti-free-radical activity observed with the Scullcap extract [38]. Also baicalin (glucuronide) efficiently scavenged superoxide radical, even 3 times more effectively than baicalein (aglycone), although it did not exert a measurable impact on scavenging of hydroxyl radical [39]. As detected by the EPR technique baicalein and baicalin form stable semiquinone radicals, which account for their radical scavenging action [36, 41]. Baicalein semiquinone radical could be further oxidized into dehydrobaicalein and the autooxidation lead to superoxide radical together with semiquinone radical. Dehydrobaicalein can be reduced back to baicalein semiquinone radical by reductants as N-acetylcysteine, and could again become autooxidized and yield to new portion of superoxide radicals. Accordingly, incubation of neonatal rat cardiomyocytes in vitro with baicalein and N-acetylcysteine caused elevated generation of superoxide and other reactive oxygen species and lead to increased cytotoxicity to cultured cells [41]. In contrast to baicalein (aglycone), baicalin (glucuronide) was resistant to autooxidation [41]. This is an important observation which proves that in addition to their strong antioxidant effect, the Scullcap flavones exert also a pro-oxidative activity, which could be revealed in the presence of reducing compounds. Thus, in general, their application together with reducing agents (N-acetylcysteine, ascorbic acid etc.) should be avoided. It can be concluded that among the flavones extracted from Scullcap root baicalein exhibits superior activity of free radical scavenging [8, 25, 29]. However, a potent antioxidant effect of the Scullcap extract is determined not only by free radical scavenging, but also by their ability to block an important step of free radical cascade both upstream and downstream of free radical. For example, a strong inhibition of the enzymes engaged in free radical generation, as iNOS, COX-2, NADPH-dependent oxidase (oxidase dependent on reduced nicotinamide adenine dinucleotide phosphate), lipoxygenases (LOX), xanthine oxidase, are well documented [34, 35, 39, 42, 43]. Mechanisms of the enzyme inhibition by the Scullcap flavones include both direct interaction/inhibition, as in the case of xanthine oxidase [36-37, 44] or NADPH: quinoneoxidoreductase [32, 42], and also a decrease of the enzyme expression [43, 45]. In lipopolysaccharideinduced inflammatory reaction in rat macrophages in vitro baicalein lead to marked decrease of iNOS and COX-2 activity, probably due to suppression of transcription activator nuclear factor–B (NFB) by the flavone in this culture system [43]. Decreasing generation of free radicals together with radical scavenging potency of baicalein, wogonin and oroxylin A account for a neuroprotective effect of



Scullcap flavones in neuronal damage in various types of oxidative stress.

balance between anti- and pro-inflammatory properties breaks down and inflammatory reactions, being detrimental to neurons, predominate [48, 49]. In this stage of the disease an antiinflammatory therapy could help to slow down a progression of neurodegeneration. However, neurodegeneration will progress; probably more slowly, and inevitably, will lead to enlargement of toxic amyloid deposits and to degeneration of neurons. Microglial cells still will be the only cells able to remove increasing amyloid deposits and debris of degenerating neurons. An anti-inflammatory therapy would be probably much more effective if it is applied together with neuroprotective therapy [49]. Drugs acting against dual targets exert both anti-inflammatory activity and neuroprotective effects could provide a valuable improvement in controlling neurodegeneration.

The Scullcap flavones could prevent deleterious effects provided to cells by oxidative stress also acting downstream of free radical cascade. This may include an inhibition of apoptosis and, in general, of cytotoxicity, and it is probable that the decrease of cell apoptosis could be also a result of inhibitory action of free radicals generated during baicalein auto-oxidation. For instance, in cultured rat cardiomyocytes oxidative stress caused by hypoxia/reoxygenation procedure was markedly decreased in cultures pretreated with baicalein (10μM), and, as a result, a significant decrease of dead cell counts was observed [41]. The cytoprotective effect was due to increased level of hydrogen peroxide, probably derived from superoxide radical generated by baicalein auto-oxidation cycle in the presence of reductants [41]. Other experiments confirmed the anti-apoptotic effect of baicalein which was strongly correlated with its auto-oxidation cycle, e.g.: human hepatoma cell line pretreated with baicalein showed an increase of hydrogen peroxide level, and hydrogen peroxide inhibited apoptosis caused by transforming growth factor--1in those cell cultures [46]. Inhibition of apoptosis is an important mechanism of cytoprotection in neurons permanently exposed to oxidative stress, thus it could be an attractive therapeutic strategy to prevent and to delay neurodegeneration. Probably, the Scullcap flavones are good candidates on medicines able to prevent apoptosis in damaged neurons; however, their anti-apoptotic effect seems to be complex, and both: anti-oxidative and pro-oxidative mechanisms could be considered. THE SCULLCAP FLAVONES ACTIVITY IN MODULATION OF NEUROINFLAMMATORY REACTIONS Inflammatory reactions in the brain tissue are believed to play a crucial role in the development and the progress of major chronic neurodegenerations. These include AD, PD, and Huntington’s disease, amyotrophic lateral sclerosis, all of the tauopathies, multiple sclerosis and many other less common conditions. Morphologically, the level of inflammation is determined by the proliferation and degree of activation of glial cells: microglia and astrocytes [47-49]. It is a commonly accepted paradigm that microglia overactivation is detrimental to the neuronal cells, by increased production and release of pro-inflammatory cytokines, free radicals, and, in general, reactive species of oxygen and of nitrogen. They lead to degeneration and to death of neurons, mainly in apoptotic manner. Degenerating neurons and presence of cell debris provide evident signal for further increase of microglial cells activity, which again enhance inflammatory cytokines and free radicals release. This self-propelling, vicious cycle between damaged neurons and dysregulated, persistent inflammation is the driving force leading to progressive neurodegeneration [49]. Therefore the anti-inflammatory drugs should be the best choice in treatment regimens of neurodegeneration. However, the failure of many clinical trials with non-steroidal anti-inflammatory drugs (NSAIDs), both specific and nonspecific inhibitors of COX’s, to slowdown a progression of neuronal degeneration have lead to new perception of the changing role of microglia in development of neurodegeneration [48, 49]. Specifically, it was stressed that microglial cells exert a potent neuroprotective activity in early stage of neurodegeneration. They are necessary for elimination of amyloid deposits by phagocytosis, release anti-inflammatory cytokines such as TGF- (transforming growth factor-), IL-10 (Interleukin-10) which are important in self-limiting of inflammatory reactions aside from release of pro-inflammatory cytokines, microglia cells are able to take up a glutamate upon stimulation with lipopolysaccharide and thereby contribute to neuronal survival [48, 49]. Therefore, the use of drugs which decrease function of microglial cell, and especially recommended doses of the drugs for prevention of neurodegeneration should be carefully reconsidered. When neurodegeneration has developed, a

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The Scullcap flavones fulfill well the criteria given above, since baicalein, wogonin and oroxylin A exert strong neuroprotective effects on degenerating neurons and also possess a marked antiinflammatory and anti-oxidative potential. Many studies report that baicalin, wogonin and oroxylin A have a strong anti-inflammatory activity due to their inhibition of the NF-B pathway [43, 50]. Anti-inflammatory activity of the Scullcap flavones are connected with their ability to inhibit expression of monocyte chemotactic protein-1, an important factor for early inflammatory responses [42, 51], and to regulate several inflammation-associated genes such as iNOS, cyclooxygenases and, thereby, inhibit production of nitric oxide (NO) and prostaglandin E2 [42, 51]. Baicalein (aglycone) and baicalin (glucoside) inhibited the inflammatory responses in vitro in peripheral blood leukocytes; a marked decrease of reactive oxygen species was noticed, inhibited myeloperoxidase (MPO) activity and also decreased expression of adhesion molecule receptors on activated leukocytes were observed [52]. In cultures of mouse microglial cells (BV-2) [53] and in rat primary microglial cells in vitro [54] baicalein markedly inhibited iNOS, thereby decreased a level of NO release. Wogonin, oroxylin A and baicalein markedly lowered the activity of iNOS, COX-2 by decreasing expression of iNOS and COX-2 genes, not by direct inhibition of the enzymes [43, 55]. Wogonin, oroxylin and baicalein are known as strong inhibitors of human lipoxygenases (LOX) [56]. In experimental mouse ischemia baicalein strongly inhibited 12/15-LOX in the ischemic brain, and also had a marked antioxidant properties [57]. In general, many authors indicate that potent anti-inflammatory activity of the Scullcap flavones correlates with their free radicalscavenging and anti-oxidant actions and, together, they are probably a main mechanism of their neuroprotective effects [34, 52, 54]. INFLUENCE OF THE SCULLCAP FLAVONES COGNITIVE FUNCTION AND MEMORY

ON

It is commonly accepted opinion, that flavonoids in general are able to improve cognitive functions and augment short- and longterm memory in experimental animals and in humans [1, 24]. Suggested mechanisms which could explain their beneficial effects include decrease of free radical levels, increase of cerebral blood flow, augmentation of neuronal cholinergic transmission and protection of the striatal dopaminergic system [1]. On the level of cellular architecture elements responsible for memory and learning beneficial actions of flavonoids are likely to stimulate formation of interneuronal connections/communications, increase of neuronal spine density and angiogenesis in hippocampal neurons (the effects described for (-)epicatechin) [7, 56]. Increase of spine density and morphology is important for synaptic plasticity in learning and memory [1, 4, 24]. On the molecular level flavonoids are able to improve long-term potentiation and enhance object recognition by mechanisms dependent on the activation of ERK 1/2 and cAMP response element-binding protein (CREB) signaling pathways,

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brain derived neurotrophic factor (BDNF), and also an activation of pro-survival PI3K/Akt signaling cascade, as was described in mice and rats treated with several flavonoids [57-59].

behavioral effects [68]. It was established that among the Scullcap flavones wogonin had the highest affinity to BZD binding site on GABA A receptor, thus exerted relatively the strongest neurological and behavioral effects [68-69]. Anticonvulsant effect of wogonin on chemically induced or electroshock seizures was established in mice and in rats [68]. Wogonin significantly blocked convulsion induced by pentylenetetrazole and by electric shock but not convulsion induced by strychnine [68]. Wogonin also exhibited relatively mild sedative and myorelaxant effects in comparison to diazepam, the standard drug from the BZD family, but was effective in axiety and motor discoordination [70, 71]. Interestingly, some authors suggested that the observed beneficial effects of the Scullcap flavone – oroxylin A- on cognitive and on mnestic dysfunctions, induced in mice by transient bilateral carotid artery occlusion, were dependent in major part on its modulation of GABA A receptor function [62, 72].

It seems likely, that Scullcap flavones share the cognitionenhancing mechanisms which are same or similar to those cited above for various flavonoids. In fact, neuroprotective effect and improvement of cognitive function with the Scullcap extract were well recognized in rodents, both in senescent animals and in the brain hypoxia model [60, 61]. Obtained results lead the authors to conclusion that the Scullcap extract could be useful in the treatment of senile dementia in humans. However, current study on individual flavones exhibited marked differences of their neuroprotective potency and revealed various mechanisms of action. In experimental animal hypoperfusion induced by transient bilateral common carotid occlusion in mice treated with oroxylin A (given orally with 1,25-5 mg/kg, 60 min. after hypoperfusion, once a day for a week) a marked improvement of memory was noticed (as compared to control animals) and the effect was correlated with the presence of phosphorylated CREB and BDNF in hippocampal neurons [62]. In the same transient hypoperfusion model in rat’s baicalein given intraperitoneally (2-4 mg/kg/day) markedly reduced cognitive deficits, and biochemical examination of brain tissue showed reduced levels of an oxidative stress markers - decrease of superoxide dismutase (SOD) activity and malondialdehyde (MDA) level [63]. In the case of wogonin its neuroprotective effect was explained mainly by strong anti-inflammatory activity [64, 65]. However, in the in vitro model of neuronal excitotoxicity wogonin did not elevate neuronal cell survival, and even enhanced glutamate-caused neurotoxicity [66]. In a recent paper [64] it is documented that wogonin induces differentiation of neural precursor cells and increased neurite outgrowth. Neural precursor cells are present in adult brains and, after stimulation, they are able to differentiate into mature neurons to replenish damaged and dead neurons. Neurite outgrowth and increase of neurite spine density are morphological structures which determine synaptic plasticity, and, as a result, cognitive function and memory. Therefore, the described effect of wogonin seems to be important for elaboration of future strategy to be used in the treatment of neuronal damage and neurodegeneration. Comparison of the available data lead us to the conclusion, that baicalein and oroxylin A exhibit potent neuroprotective effect accompanied with cognitive function improvement and enhancement memory, whereas wogonin reveals a marked neuroregeneratory potency. Even if some authors announce that wogonin could increase the glutamate neurotoxicity, and the effect remains to be precisely evaluated, current human use of Scullcap crude extract seems to be safe, since the wogonin content in the extract is very low (about 20 fold lower than baicalein). However, an attractive future strategy aimed to regenerate neuronal tissue in brain damage and neurodegenerative diseases, will need an application of higher doses of pure wogonin (currently available is synthetic wogonin), and in this case wogonin should be used together with glutamate receptor-inhibitors. ANTICONVULSANT AND AXIOLYTIC EFFECTS OF THE SCULLCAP FLAVONES Flavonoid-rich extract from Scullcap root exhibited various beneficial neurological and behavioral effects in laboratory animals (rodents) as anticonvulsive, anxiolytic, myorelaxant and sedative activities [67]. It was documented that flavones isolated from Scullcap roots act as allosteric positive modulators on the inhibitory effects of GABA A (-aminobutyric acid A- receptor) at the benzodiazepine (BZD) binding site on the GABA A receptor complex [68-70]. They increased the frequency of chloride channel openings of the receptor, enhanced the inhibitory effect of GABA, and that mechanism of action accounted for their neurological and

It can be summed up that baicalein, wogonin, oroxylin A exhibit favorable impact on behavior: marked anxiolytic effect, mild sedative, motor coordination improvement as well as moderate anticonvulsive activity. These effects of the flavones should be applied to long term prevention of epileptic seizures and also could be used in complementary therapy of neurodegenerative diseases. IMPACT OF THE SCULLCAP FLAVONES ON AMYLOIDOGENIC PROTEIN WITHIN THE BRAIN Aggregation of -synuclein, an abundant and conserved presynaptic protein, is implicated as a critical factor in several neurodegenerative disease such as PD, dementia with Lewy bodies, variant of AD with Lewy bodies, multiple system atrophy [73]. Natively -synuclein is unfolded protein and its structure strongly depends on local microenvironment and it is able to accommodate a number of conformations and form neurotoxic aggregates. Currently there is no available therapeutic agent able to prevent the protein aggregation, therefore the search for such agents includes extensive screening studies of plant- derived active compounds, especially flavonoids. Impact of 48 common flavonoids from several classes (mainly baicalein) on aggregation/disaggregation of -synuclein in bacterial cells transfected with human recombinant -synuclein-containing plasmid pRK172 was evaluated [74]. It was established that several flavonoids, among them baicalein, strongly inhibited fibrillation, and aggregation of existing fibrils [74]. Importantly, baicalein-synuclein complexes, i.e. baicalin-stabilized oligomers of the protein, were not cytotoxic to cells of human dopaminergic neuroblastoma line SH-SY5Y [75]. In this study it was found that many compounds that inhibited -synuclein aggregation also inhibited the formation of amyloid- and of tau protein filaments [75]. Analysis of the data presented in this paper showed that among 79 of tested natural and synthetic compounds baicalein did belong to a group of the strongest inhibitors of amyloidosis; it effectively decreased formation of -synuclein, amyloid- and tau protein filaments [75]. Chemical studies on the nature of baicalein--synuclein interactions showed that baicalein in its oxidized form (semiquinone) disaggregated existing fibrils and formed a complex with soluble oligomer of -synuclein, in which the protein was covalently modified by baicalein quinone to form a Schiff base with a lysine side chain in -synuclein [76]. In cell models of familiar Parkinsonism [77] baicalein reduced fibrillation of -synuclein with a concentration-dependent decrease in beta sheet conformation and rescued toxicity of synuclein aggregates to mouse neuroblastoma N2A cells. Baicalein also protected rat cortical cells from amyloid- (25-35)–induced apoptosis, probably by inhibiting 12-lipoxygenase mediatedactivation of c-jun dependent apoptosis [78], and both baicalein and baicalin were equally potent inhibitors of amyloid- induced neurotoxicity to rat pheochromocytoma PC12 cell line [79-81]. The beneficial influence of baicalein on neurotoxic effects of amyloid- was also confirmed in laboratory animals. Intraperitoneal


injection of amyloid- (25-35) peptide caused amnesia in mice and baicalein strongly attenuated the signs of amnesia [42]. It is possible that other flavones present in Scullcap root exhibit anti-amyloidogenic activity also in vivo. For instance, it was observed that baicalein markedly ameliorated memory impairment induced in mice by intracerebroventricular injection of amyloid(25-35) [80]. The beneficial effect of oroxylin A on the memory impairment was mediated by GABAergic transmitter system (single administration of oroxylin A) and by lowering of astrocyte and microglia activation, decrease of free radicals generation and increase of cholinergic transmission (prolonged administration of oroxylin A) [81]. Also other flavones from Scullcap roots, aglycones and glucuronides should be tested in the aspect of their influence on amyloidogenic processes in neurodegeneration. CONCLUSIONS The results in the papers cited above show that baicalein, and probably other Scullcap flavones are able both to prevent fibrillation/aggregation of amyloidogenic proteins and, to disaggregate of existing protein deposits in vitro. Baicalein and oroxylin A exhibit potent neuroprotective effect, improve cognitive function and enhance memory in brain hypoperfusion models in laboratory animals, whereas wogonin reveals a neuroregeneratory potency in primary cultures of neurons. The Scullcap flavones exert potent anti-inflammatory activity which correlates with their free radical-scavenging and anti-oxidant actions and, together, they constitute a main mechanism of their neuroprotective effects.


[7]

[8]

[9] [10]

[11] [12]

[13]

[14]

[15] [16]

ACKNOWLEDGEMENT This study was supported by the Polish Academy of Sciences.We are very grateful for Ms. Galina Alieva and her editorial works throughout the preparation of this manuscript.

[17]

[18]

ABBREVIATIONS AD

=

Alzheimer Disease

COX

=

Cyclooxygenase

IL

=

Interleukin

iNOS

=

Inducible Nitric Oxide Synthase

GABA

=

Gamma-Butyric Acid

NF-KB

=

Nuclear Factor-KappaB

NO

=

Nitric Oxide

PD

=

Parkinson disease.

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Revised: September 28, 2010

Accepted: November 17, 2010