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Journal of the American College of Nutrition, Vol. 26, No. 4, 341-349 (2007)
Published by the American College of Nutrition

Short-Term Supplementation with Plant Extracts Rich in Flavonoids Protect Nigrostriatal Dopaminergic Neurons in a Rat Model of Parkinson's Disease

Krishna P. Datla, PhD, Virginia Zbarsky, PhD, Deepal Rai, BSc, Shabnam Parkar, BSc, Naomi Osakabe, PhD, Okezie I. Aruoma, DSc and David T. Dexter, PhD

Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Faculty of Medicine, Imperial College, Charing Cross Campus (K.P.D., V.Z., D.R., S.P., D.D.)
The Food Research Centre, Department of Applied Science, London South Bank University (O.I.A.), London, UNITED KINGDOM
Meiji Seika Kaisha Limited, Food and Health R & D Laboratories (N.O.), Saitama, JAPAN
Department of Veterinary Public Health, College of Veterinary Medicine, Seoul National University (O.I.A.), Seoul, KOREA

Address reprint requests to: Dr. David T. Dexter, Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Faculty of Medicine, Imperial College, Charing Cross Campus, St. Dunstan's Road, London W6 8RP, UNITED KINGDOM. E-mail: d.dexter{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: Antioxidants from plants were known to reduce the oxidative stress by scavenging free radicals, chelating metal ions and reducing inflammation. As increased oxidative stress was implicated in the nigrostriatal dopaminergic neuronal loss in Parkinson's disease (PD), we have assessed whether the plant extracts protects the nigrostriatal dopaminergic neurons in the animal model of PD.

Methods: Male adult Sprague-Dawley rats were treated orally between 10am–11am each day with the extracts from tangerine peel, grape seeds, cocoa and red clover for four days. One hour after the final dosing, the left medial forebrain bundle was lesioned by infusing the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA; 12µg) under anaesthesia. Seven days post-lesion, the number of dopaminergic cells in the substantia nigra pars compacta and the levels of dopamine and its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striata were quantified and compared with the vehicle-treated groups.

Results: Compared to the unlesioned side, 6-OHDA lesions significantly reduced the number of dopaminergic cells and the levels of dopamine and its metabolites DOPAC and HVA in the vehicle-treated animals. Pretreatment of animals with extracts of tangerine peel (rich in polymethoxylated flavones; 35 mg/kg/day), cocoa-2 (rich in procyanidins; 100 mg/kg/day) and red clover (rich in isoflavones; 200 mg/kg/day) significantly attenuated the 6-OHDA-induced dopaminergic loss. However, no significant protection was seen in animals supplemented with red and white grape seeds (rich in catechins; 100 mg/kg/day), and cocoa-1 (rich in catechins; 100 mg/kg/day).

Conclusions: Pre-treatment of plant extracts rich in polymethoxylated flavones, procyanidins and isoflavones but not catechins protected the nigrostriatal dopaminergic neurons in the rat model of PD.

Key words: ntioxidants, flavonoids, 6-hydroxydopamine, neuroprotection, Parkinson's disease


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Parkinson's disease (PD) is a progressive neurodegenerative disease affecting about 1.6% of the general population aged over 65 [1] and the number of PD patients is predicted to rise as life expectancy increases. The clinical symptoms of PD, manifest as the loss of initiation and control of movement and appear only after a substantial loss of dopaminergic neurons (50–60%) in the substantia nigra pars compacta (SNpc) in the midbrain [2]. As a consequence of the cell loss, dopamine in the terminal region of striata is depleted by 70–80% [3].

Several mechanisms have been proposed to contribute to the loss of neurons in PD with increased oxidative stress being strongly implicated [4,5]. Oxidative stress-induced cell death is mediated by the increased activity of highly reactive oxygen species (ROS) such as superoxide and hydroxyl radicals, and the reactive nitrogen species (RNS) such as peroxynitrite radicals. In healthy cells ROS and RNS generated as by-products of cellular metabolism and mitochondrial respiration are neutralized by the cellular antioxidant defense mechanisms. Cells neutralize free radicals by enzymatic and non-enzymatic mechanisms. Antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase and non-enzymes antioxidants such as reduced glutathione, ascorbic acid, and {alpha}-tocopherol are key components of the cellular defense [6]. The detection of substantially high levels of oxidized lipids [7,8], proteins [9,10] and nucleic acids in postmortem PD brains [11,12] suggests that the cell death in PD could be a consequence of increased generation of free radicals and/or decreased cellular antioxidant defenses. Epidemiological studies show that the incidence of PD was inversely associated with the intake of diet rich in the natural antioxidant Vitamin E [13,14]. Unfortunately, results from the clinical trials using Vitamin E in PD were conflicting. The supplementation of 2000 IU dl-{alpha}-tocopherol per day was found to be ineffective in slowing down the disease progression [15] while another study, using a supplement consisting of a high dose of {alpha}-tocopherol (3200 IU) and Vitamin C, was shown to be effective in delaying the need for L-DOPA by up to 2.5 years in drug naive patients [16]. The latter results suggest that use of antioxidant supplements can be beneficial to PD patients as they slow down the progression of the disease. Recently, ropinirole (a dopamine D2/D3 receptor agonist) treatment was shown to slow the progression of PD [17]. The precise mechanisms by which dopamine agonists protect the neurons are far from clear but may involve antioxidant mechanisms [18]. Unfortunately, prolonged supplementation of these agents is associated with long term side effects. Hence, there is an urgent need for the development of alternative neuroprotective strategies for the treatment of PD [19].

Leaves of green tea, peels and seeds of grapes and seeds of cocoa are rich in flavonoids [20–23]. Flavonoids have antioxidant and several other functions and may limit oxidative damage in vitro by inhibiting the activities of lipoxygenases [24,25], directly scavenging free radicals [26–29], chelating metal ions [29], decreasing the influx of Ca2+ [27], increasing cellular antioxidant levels [27,30] and reducing inflammation [22,31–34]. Given the involvement of oxidative stress, increased iron levels and inflammatory mechanisms in PD, it is of interest to see whether the mixed function nature of flavonoids could offer neuroprotection. Indeed, a 4 day treatment of tangeretin, a flavonoid from tangerine peel, significantly protected nigrostriatal dopaminergic neurons in the 6-OHDA-lesion model of PD in our earlier study [35]. In this study we have examined the neuroprotective effects of antioxidant-rich plant extracts, namely tangerine peel (tangeretin 60% and nobiletin 40%), cocoa-1 (catechins 3%) cocoa-2 (procyanidins 6.5%), red and white grape seeds (catechin monomers 8%, polymers 65% and oligomers 27%) and red clover (formononetin 26%, biochanin A 12%, diadzein 1.5% and genistein 1%) (Fig. 1), were assessed using the 6-OHDA-lesion rat model of PD.


Figure 1
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  		Fig. 1. Structures of main flavonoids present in the extracts.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Materials
Fresh extract solutions were made each day just before use. The powders of tangerine peel extract (Department of Citrus, Florida, USA) and red clover extract (Linnea SA, Switzerland) were made into a paste with Cremophor (3% of total volume; Sigma, UK) followed by the addition of sterile water to make up the volume. Cocoa extracts (Meiji, Saitama, Japan) solutions were made similarly but ethanol was used instead of Cremophor. Solutions of grape seed extracts (Polyphenolics Inc, USA) were made in sterile water. Neurotoxin 6-hydroxydopamine (6-OHDA) and all other chemicals used were purchased from Sigma-Aldrich (Dorset, UK). Polyclonal tyrosine hydroxylase (TH) antibody was obtained from Chemicom (Harrow, UK), and the ABC immunostaining kit from Vector Laboratories (UK).

Animals
Adult male Sprague-Dawley rats (Harlan, UK) weighing 200–215 gm were used in the study. The rats were housed in groups of three animals per cage in standard animal holding facilities (maintained at temperature 21° ± 1 °C; 12h light-dark cycle; 0700 hrs lights on) with free access to drinking water and pellet diet (rat standard diet; SDS, U.K.). Animals were handled daily and allowed one week to acclimatize to the conditions of animal holding facilities before starting any procedure. All scientific procedures were carried out with the approval of the Home Office, UK.

Unilateral Partial Lesioning of the Medial Forebrain Bundle with 6-OHDA
For each extract study 12 animals were used. Six animals received vehicle and the remaining six received extract treatment. To reduce any interference with the absorption of the extracts, each day animals were dosed between 10am–11am, when the food intake was minimal, by gavage for 4 days. The doses of extracts were: tangerine peel (35 mg/kg/day), cocoa-1 (100 mg/kg/day) and cocoa-2 (100 mg/kg/day), red and white grape seeds (100 mg/kg/day) or red clover (200 mg/kg/day). The dose of each extract selected was based on either our own studies [35, Datla et al unpublished] or from the literature. One hour after the final dosing, left medial forebrain bundle (stereotactic co-ordinates: 2.2 mm posterior, 1.5 mm lateral from bregma and 7.9 mm ventral to dura with ear bars 5 mm below incisor bars; [39]) was lesioned with 6-OHDA (12µg) as previously described [35] under isoflurane anaesthesia. One week after surgery, the animals were killed by cervical dislocation, brains rapidly removed and cut at the level of the infundibular stem to separate the hindbrain block (containing the SNpc) and the forebrain block (containing the striata). From the forebrain left (lesioned) and right (unlesioned) striata were dissected out and stored separately at –80°C until analysis.

Quantification of Nigral Dopaminergic (Tyrosine Hydroxylase Positive) Cells: Immunohistochemistry
The hindbrain block containing the nigra was fixed in 4% paraformaldehyde for 7 days, and cryoprotected in 30% sucrose solution before rapid freezing and storing at –80°C. From the frozen block, 20 µm thick sections were cut coronally using a refrigerated cryostat (Bright, UK). Immunostaining for tyrosine hydroxylase (TH; rate limiting enzyme for the synthesis of dopamine) was utilized to identify dopaminergic neurons, as described previously [40]. Briefly, the free-floating sections were rinsed with phosphate-buffered saline (PBS) followed by blocking with 20% normal goat serum (ICN Pharmaceuticals, UK) in PBS for an hour on a shaking platform. After rinsing with PBS, the sections were incubated with polyclonal rabbit anti-TH antibody (1:3000; Chemicon, UK) over night at room temperature. On the following day, after incubating the sections with biotinylated anti-rabbit IgG (Vector Laboratories, UK), the TH-antibody complex was visualized with 3-3'-diaminobenzidine and H2O2. TH +ve cells were visualized under bright field illumination using a Nikon Eclipse E800 microscope and counted manually. Minimum of three sections were counted at level B (–5.1 mm from bregma) per animal in the SNpc according to Carman et al 1991 [39] and an average number of dopaminergic cells was taken. At level B the area of SNpc is very well defined and easy to identify as the medial terminal nucleus of the accessory optic tract, where no cells are stained, clearly separating the SNpc from the ventral tegmental area thus allowing unbiased counting of this small number of cells. Earlier studies have used this region for consistency when comparing the cells across different treatment groups [39].

Quantification of Striatal Dopamine and its Metabolites DOPAC and HVA: HPLC Analysis
Striatal dopamine and its metabolites DOPAC and HVA were quantified by HPLC-ECD as described previously [39]. Briefly, unlesioned and lesioned striata were individually weighed and homogenized in ice-cold buffer (50 mM trichloroacetic acid, 0.5 mM, EDTA, 0.5 pmol/µl 3,4-dihydroxybenzylamine as an internal standard). The homogenates were centrifuged and the supernatants were separated, filtered (0.45µm, Whatman, U.K.) and loaded onto a temperature-controlled (5°C) auto-sampler (Gynkotek, U.K.) connected on-line to the HPLC system. Dopamine and its metabolites were separated on an Altex 3µm ODS column (4.6 mm x 75 mm, Beckman, U.K.) using a mobile phase of composition: 0.1 mM KH2PO4, 0.1 mM EDTA, 1 mM octyl sodium sulfonate, 10% methanol V/V (pH 2.75 adjusted with orthophosphoric acid). The separated components were oxidized in an analytical cell (electrode 1 set at –0.20 V and electrode 2 set at +0.34 V; ESA, UK) connected to a Coulochem-II detector (ESA, UK). The chromatograms were collected on-line and quantified automatically using Chromeleon software (Dionex, UK). Dopamine turnover was calculated using dopamine, DOPC and HVA concentrations (DOPAC+HVA/DA).

Statistical Analysis
The effects of lesion on the number of dopaminergic cells, dopamine, DOPAC, HVA and DA-turnover in each treatment group were measured by comparing the values on the lesioned side with the values on the unlesioned side using paired Student's t-test using statistical package SPSS (version 10). The percentage loss of dopaminergic cells or dopamine and its metabolites on the lesioned side was calculated from the unlesioned side for each animal and averaged for the vehicle or extract-treated group calculated. The neuroprotective effects of the extracts were evaluated by comparing the mean percentage loss of the extract-treated group with the corresponding vehicle-treated group, using unpaired Student's t-test. P values less than 0.05 were taken as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
None of the extract treatments affected the weight or behaviour of the animals compared to vehicle treated group. Similarly none of the treatments showed any significant effects on the number of dopaminergic cells in the SNpc or striatal dopamine levels on the unlesioned side. In the vehicle treated animals, lesioning of the medial forebrain bundle with 12µg of 6-OHDA significantly reduced the number of dopaminergic cells (Fig. 2a) compared to the unlesioned side (Fig. 2b). The percentage reductions of dopaminergic cells was approximately 50% in the water and Cremophor-vehicle groups (Fig. 3c, 3a, 3d) and was approximately 75% in the ethanol-vehicle group (Fig. 3b). The 6-OHDA-lesions also significantly reduced the levels of dopamine, DOPAC and HVA in vehicle treated groups. The percentage dopamine loss was very similar to the percentage loss seen with dopaminergic cells, and ranged from 50% (water group: Fig. 4c) to 90% (ethanol group; Fig. 4b).


Figure 2
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  		Fig. 2. Images of the dopaminergic cells in the substantia nigra pars compacta on the lesion side (a) and unlesioned side (b) of vehicle treated animals and on the lesioned side of animals treated with the extracts of tangerine peel (c), cocoa-2 (d), red clover (e), white (f) and red (g) grape seeds and cocoa-1 (h).

 

Figure 3
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  		Fig. 3. Percentage loss of dopaminergic cells in the substantia nigra pars compacta (SNpc) on the lesioned side compared to the unlesioned side in unilateral 6-OHDA (12µg) lesioned rats. Columns represent the mean ± SEM values. n = 6 per group. In the vehicle treated animals there were 135 ± 13 dopaminergic cells in the unlesioned SNpc. Open columns represent respective vehicle treated group. a) tangerine peel (filled column), b) cocoa-1 (vertical lines) and cocoa-2 (horizontal lines), c) red grape seed (backslash) and white grape seed (forward slash) and d) red clover (squares). *P < 0.05, **P < 0.005 vs. vehicle-treated group (unpaired Student's t-test).

 

Figure 4
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  		Fig. 4. Percentage loss of striatal dopamine on the lesioned side compared to the unlesioned side in unilateral 6-OHDA (12µg) lesioned rats. Columns represent the mean ± SEM values. n = 6 per group. In the vehicle treated animals, the dopamine levels in the unlesioned striata were 13.8 ± 0.5 ng/mg. Open columns represent respective vehicle treated group. a) tangerine peel (filled column), b) cocoa-1 (vertical lines) and cocoa-2 (horizontal lines), c) red grape seed (backslash) and white grape seed (forward slash) and d) red clover (squares). *P < 0.05, **P < 0.01 vs. vehicle-treated group (unpaired Student's t-test).

 
The percentage loss of dopaminergic cells after 6-OHDA lesions was significantly attenuated in animals pretreated with extracts of tangerine peel (Fig. 2c; 3a, P<0.05), cocoa-2 (Fig. 2d; 3b, P<0.02) and red clover (Fig. 2e; 3d, P<0.05) but not in either of the grape seed extract or cocoa-1 treated groups (Fig. 2f, 2g and 2h; 3b and 3c), compared to the percentage loss of vehicle-treated groups. Correspondingly, the percentage loss of dopamine was significantly attenuated in the animals pretreated with tangerine peel extract (Fig. 4a, P < 0.005), cocoa-2 (Fig. 4b, P < 0.05) and red clover extract (Fig. 4d, P < 0.05) compared to the respective vehicle groups. Neither white or red grape seed extracts, nor cocoa-1 extracts prevented the loss of dopamine (Fig. 4b, 4c). 6-OHDA-lesion also reduced the concentrations of DOPAC and HVA significantly in the vehicle-treated animals (Table 1). And in animals treated with tangerine peel extract and red clover, DOPAC levels were significantly augmented compared to the respective vehicle groups (P < 0.01 vs. lesioned side of the vehicle group respectively; Table 1). In the other extract treated groups, concentrations of DOPAC or HVA on the lesion side were not significantly different from the corresponding vehicle treated groups. No significant changes in dopamine turnover were observed on the lesioned side in vehicle or extract treated groups (data not shown).


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  		Table 1. Effects of Pretreatment of the Extracts on the Striatal Dopamine Metabolites DOPAC and HVA in 6-OHDA Lesioned Rats

 

   DISCUSSION
 
This study reports that the supplementation of rats with extracts rich in tangeretin and nobiletin (from tangerine peel), procyanidins (cocoa), isoflavones (red clover) protected the nigrostriatal dopaminergic neurons in 6-OHDA lesion model of PD. Surprisingly, pretreatments of catechin rich extracts from grape seeds and cocoa failed to protect the dopaminergic neurons, at least at the dose and supplementation schedule used in this study.

Unilateral lesioning of the medial forebrain bundle of rats with 6-OHDA is a commonly used animal model [41] as it reflects both the nigrostriatal dopaminergic loss and increased oxidative stress of PD [42]. The dopaminergic neurons die as a consequence of increased oxidative stress with 6-OHDA infusions. Auto-oxidation of 6-OHDA [43,44] was shown to generate toxic quinines [45], superoxide radicals, hydrogen peroxide, and hydroxyl radicals [46]. Other sources for the ROS are dysfunctional mitochondria or Fenton reactions as 6-OHDA also inhibit mitochondrial respiration [47] and increases the availability of reactive iron by releasing it from the ferritin [48,49]. 6-OHDA also activates glia [50] which can release ROS and RNS and inflammatory cytokines [51].

The tangerine peel extract used in this study is composed of tangeretin (60%) and nobiletin (40%). Earlier, we have shown that tangeretin crosses the blood brain barrier (BBB) after oral administration in rats and that the distributions in the brain was up to 4 times higher than the peripheral organs [35]. There are no direct studies indicating that nobiletin crosses the BBB. However, other studies have demonstrated that nobiletin is absorbed from the gut in rats after oral administration [52]. Also, with 6 methoxy groups, nobiletin should be more lipophilic than tangeretin (with 5 methoxy groups; Fig. 1a) and therefore should be more likely to cross the BBB than tangeretin.

Tangeretin pretreatment (20 mg/kg/day x 4) was also shown to protect dopaminergic cells in the SNpc and dopamine levels in the striata [35]. In this study, with the use of 35 mg/kg of tangerine peel extract (i.e., 23.3 mg of tangeretin and 11.7 mg of nobiletin; a dose of tangeretin comparable to the earlier study), showed similar neuroprotection. As the level of protection seen here was not any better than in the previous study, this may suggest that it is unlikely that tangeretin and nobiletin were acting synergistically.

Structure-activity relationship studies suggest that flavonoids can have antioxidant properties by scavenging the free radicals and chelate the metal ions. Thus, flavonoids with a C2–C3 unsaturation (see Fig. 1), a catechol moiety in the rings A or B, or a flanking hydroxyl groups at C3 and A5 and C4-oxo group will be able to scavenge the hydroxyl radicals and chelate the iron [24,53–55]. Based on these properties, tangeretin and nobiletin should have antioxidant properties as they have a C2–C3 un-saturation and C4, A5 polar groups that can scavenge the hydroxyl radicals and chelate iron.

Grape seeds and cocoa contains large quantities of (+) catechin, (–)-epicatechin, (–)-epicatechin-(3-O-)-gallate and procyanindins (oligomeric or polymeric flavan-3-ols; [56,57]). These extracts are known to inhibit lipid peroxidation [58,59], scavenge free radicals [20,60], and inhibit inflammation [31,61–63]. The treatment of cocoa-1 and both red and white grape seed extracts (rich in catechins) did not show any significant neuroprotection, while cocoa-2 treatment (rich in procyanidins) was effective. Supporting our findings with catechins, a recent study demonstrated that a 2-week grape seed phenolic treatment did not increase the plasma free radical scavenging activity albeit, in hamsters [64]. It is important to point out that in order for compounds to have any neuroprotective effects, they must be able to cross the BBB. The lack of neuroprotective effects seen here cannot be attributed to a lack of transportation across the BBB, on the contrary, they were known to cross the BBB [65]. Paradoxically, neuroprotection seen with the procyanidins is difficult to explain because of their high molecular weights, their absorption from the gut seems negligible [58,63]. In another study procyanindins dimers were detected in the plasma in a nanomolar range [67]. Thus we speculate that either minute amounts of procyanidins or that their biologically active metabolites [68] may be involved in the neuroprotection.

There was a greater loss of dopaminergic cells and dopamine levels in the ethanol based vehicle and cocoa groups compared to the other vehicle groups. Studies on rats fed ethanol demonstrate increased lipid peroxidation, TNF-{alpha} expression, necrosis and mild-inflammation in the brain [69,70]. The pronounced damage seen in the ethanol treated animals of this study could be a consequence of synergistic effects of 6-OHDA and ethanol. Indeed, a recent in vitro study supports such synergistic effects [71].

Catechins were not able to protect the dopaminergic neurons from 6-OHDA-toxicity. Catechins have a C3-hydroxyl group and a catechol moiety on the ring B, and they lack C4-oxo or C2–C3 unsaturation (Fig. 1). Thus, presence of a C4-oxo and or a C2–C3 unsaturation seems to be critical for the neuroprotection, especially in this model. Indeed, red clover extracts protected the dopaminergic neurons and are rich in isoflavones with a C4-oxo group and C2–C3 double bond. Also the presence of hydroxyl groups at 3 and 3', a feature of 17ß-estradiol and related estrogens, could play a significant role in the neuroprotection [72]. Recently, red clover extract treatment was shown to increase the uterine weight, vaginal cell cornification and mammary gland duct branching in overectomised female rats, suggesting the estrogenic activity of isoflavones [73]. Evidence also comes from our earlier studies, where we have shown that estrogen protects the nigrostriatal dopaminergic neurons from 6-OHDA-toxicity [74,37].

Inflammation also plays an important role in the neurodegeneration in PD. Activation of microglia was consistently observed in PD brains [75,76]. The activated microglia is the source for ROS, RNS and inflammatory cytokines. As mentioned earlier, 6-OHDA was also shown to increase the microglia in rats. Therefore the neuroprotective properties of extracts in this study could also involve suppression of inflammatory cytokines. Indeed, treatment of polymethoxylated flavones such as tangeretin and nobiletin was demonstrated to inhibit lipopolysaccaride (LPS)-induced cytotoxic cytokine TNF-{alpha} expression in human monocytes [77]. Similarly, nobiletin was shown to inhibit the LPS-induced nitric oxide and superoxide generation, suppress the 12-O-tetradecanoylphorbol-13-acetate-induced expression of COX-2 and iNOS and decrease the prostaglandin E2 release in mouse macrophages and skin [78]. Anti-inflammatory activities of genistein have also reported using in vitro studies. For example, genistein was shown to suppress LPS-induced TNF-{alpha} expression in alveolar macrophages [79] and IL-2 in peripheral blood mononuclear cells [80]. Whether the neuroprotection seen with these flavonoids in our study also involves the suppression of the release of gliosis mediated-inflammatory factors remain to be elucidated.

In conclusion, animals pretreated with the plant extracts rich in antioxidant flavonoids, the nigrostriatal dopaminergic neurons were significantly protected from the 6-OHDA-induced toxicity. This study also suggests that compounds with a C4-oxo and flanking hydroxyl groups, C2–C3 double bond, or hydroxyl groups at 3 and 3' in their structure are affective neuroprotective antioxidants in the 6-OHDA model of Parkinson's disease.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank the UK Parkinson's Disease Society for the financial support. Professor Aruoma acknowledges the "Brain Pool Award" from the Korean Ministry of Science and Technology (2004–2005) and its hosting by the Seoul National University, College of Veterinary Medicine, Seoul, Korea.

Received April 4, 2005. Accepted July 21, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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