Neuroprotective Effects of a Standardized Extract of Diospyros kaki Leaves on MCAO Transient Focal Cerebral Ischemic Rats and Cultured Neurons Injured by Glutamate or Hypoxia

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Plant material
• Extraction and isolation
• LC-DAD-MS analysis
• Quantification of the flavonoids in NXQ
• Animal tests
• MCAO animal preparation
• Drugs and chemicals
• In vitro test
• Protection against glutamate toxicity in primary rat hippocampal neuronal culture
• Cell viability
• Hypoxia test in the primary rat cerebral cortical neuronal culture
• Statistical analysis
• Supporting information
• Results
• Discussion
• Acknowledgments
• References

Abstract

Naoxinqing (NXQ, a standardized extract of Diospyros kaki leaves) is a patented and approved drug of Traditional Chinese Medicine (TCM) used for the treatment of apoplexy syndrome for years in China, but its underlying mechanism remains to be further elucidated. The present study investigates the effects of NXQ against focal ischemia/reperfusion injury induced by middle cerebral artery occlusion (MCAO) in rats and against glutamate-induced cell injury of hippocampal neurons as well as against hypoxia injury of cortical neurons. Oral administrations of NXQ at 20, 40, 80 mg/kg/day for 7 days (3 days before MCAO and 4 days after MCAO) significantly reduced the lesion of the insulted brain hemisphere and improved the neurological behavior of the rats. In primary rat hippocampal neuron cultures, treatment with NXQ at 5 - 20 μg mL concentration protects the neurons against glutamate-induced excitotoxic death in a dose-dependent manner. In primary rat cerebral cortical neuron cultures, pretreatment with 5 - 100 μg/mL NXQ also attenuates hypoxia-reoxygen induced neuron death and apoptosis in a dose-dependent manner. These results suggest that NXQ significantly protects the rats from MCAO ischemic injury in vivo and the hippocampal neurons from glutamate-induced excitotoxic injury as well as cortical neurons from hypoxia injury in vitro by synergistic mechanisms involving its antioxidative effects.

Abbreviations

NXQ:Naoxinqing

CNS:central nervous system

MCAO:middle cerebral artery occlusion

I/R:ischemia and reperfusion

Introduction

Naoxingqing (NXQ, a standardized extract of Diospyros kaki leaves) is a patented and approved drug of Traditional Chinese Medicine. It has been used for the treatment of stroke or apoplexy syndrome in China to improve the outcome of ischemia stroke for years [1]. The remedy has been reported to possess superior efficacy and few side-effects in the patients who suffer from cerebral atherosclerosis, transitory ischemia syndrome, cerebral thrombo-embolism, cerebral thrombosis sequelae, and apoplexy sequelae [1], [2], [3].

Previous studies demonstrated that NXQ (30 mg/kg) could increase coronary blood flow, reduce coronary resistance in anesthetized dogs, and antagonize the main artery constriction induced by potassium chloride in isolated rabbit aortic vessels, and improve the overall circulation and cardiac function by lowering the myocardial oxygen consumption [3], [4], [5]. NXQ could also increase cerebral blood flow in anesthetized dogs [2]. Besides, it could also significantly increase the electrophoretic mobility of erythrocytes, lower whole blood and plasma viscosity, and decrease the deposit of fibrinogen in rabbits [6]. We previously reported that pretreatment of NG108-15 cells with NXQ attenuated H₂O₂-induced cell injury and apoptosis by improving redox disequilibrium under exposure of H₂O₂ as indicated by the increase in activities of intracellular endogenous antioxidants, glutathione, glutathione peroxidase and catalase, and by the decrease in the leak of lactate dehydrogenase and the accumulation of malondialdehyde [7], [8]. Despite these observations and some pharmacological research, the precise mechanisms for the efficacy of this drug on stroke are yet to be elucidated.

In recent years, it has became clearer that reactive oxygen species (ROS) oxidative stress and excitatory amino acids induced excitotoxicity are closely related to the common pathological mechanism in acute and chronic CNS injury and neurodegenerative diseases such as stroke and Alzheimer's disease (AD) [9], [10], [11]. High consumption of oxygen and lack of endogenous radical scavengers and antioxidants in the brain make the central nervous system (CNS) much more susceptible to ROS oxidative stress and excitatory amino acid toxicity. Antioxidative action and antiexcitotoxicity may protect neurons from necrosis and apoptosis induced by oxidative stress and excitotoxicity, and therefore delay or prevent the pathological process of neuronal injury and neurodegeneration in CNS. Hence this might be a reasonable therapeutic strategy for stroke and other neurodegenerative diseases [12]. Ginkgo extract is widely used as a proven natural product to treat cerebral ischemic disorders and neurological disorders [13]. We here used Ginkgo extract as a reference to examine the neuroprotection of NXQ in rat against ischemia and reperfusion (I/R) injury and in primary neuronal culture against oxidative stress injury.

Materials and Methods

Plant material

Leaves of Diosypros kaki L. were collected in Gongchen county, Guangxi Procvince, China, in October 2002, and identified by Professor Wen-Bo Liao, Hortus Siccus, Sun Yat-sen University.

Extraction and isolation

NXQ, supplied by Hutchison Whampoa Guangzhou Baiyunshan Chinese Medicine Ltd., was prepared according to the following procedure. Air-dried leaves of D. kaki (10.0 kg) were extracted with boiling water. The extract was concentrated and 95 % ethanol added to make 70 % ethanol in solution. The collected supernatant was evaporated to dryness under vacuum, and then extracted with ethyl acetate. After evaporation, concentration and drying, the ethyl acetate extract (NXQ, about 220 g) was collected.

LC-DAD-MS analysis

The HPLC fingerprint of NXQ was obtained from LC-MS with an LCQ-DECA XP liquid chromotograph-mass spectrometer unit (Thermo Finnigan).

HPLC conditions: Waters Symmetry Shield ^TM RP₁₈ column (5 μm, 4.6 × 250 mm) at a column temperature of 25 °C; gradient system of (A) 0.2 % HOAc, (B) MeOH and (C) CH₃CN: 0 - 5min, 75 - 60 % A, 10 - 35 % B, 15 - 5 % C; 5 - 25 min, 60 - 50 % A, 35 - 45 % B, 5 % C; 25 - 30 min, 50 - 45 % A, 45 - 50 % B, 5 % C; 30 - 45 min, 45 - 50 % A, 50 - 45 % B, 5 % C; 45 - 55 min, 50 - 75 % A, 45 - 10 % B, 5 - 15 % C; Flow rate: 0.5 mL/min; injection volume: 10 μL. Sample concentration: 1 mg/mL in MeOH (filtered with Millipore filter before LC-MS assays).

DAD conditions: 254 nm. Negative-ion ESI-MS was measured by a full range scan from 150 - 1000 units with source temperature: 350 °C, ion spray voltage 4.0 kV and de-clustering potential: 20 V.

Quantification of the flavonoids in NXQ

The extract was dissolved in HPLC grade MeOH at a concentration of 5 mg/mL, then 36 % HCl was added to the solution to finally reach 2 M in HCl. The mixture was hydrolyzed at 90 °C for 2 hours. Then the solution was diluted with MeOH to a concentration of 0.5 mg/mL, filtered with a Millipore filter before HPLC assays. HPLC conditions: Waters Symmetry Shield ^TM RP₁₈ column (5 μm, 4.6 × 250 mm) at column temperature of 25 °C; solvent system: MeOH-0.4 %H₃PO₄ (50 : 50); flow rate: 1.0 mL/min; injection volume: 10 μL and a UV detector at 360 nm. A calibration curve was made with 2, 5, 10, 20, 30 μg/mL of quercetin and kaempferol in MeOH. The content of flavonoids in NXQ was calculated as in the following formula by using the areas of absorbtion peaks of quercetin and kaempferol in the sample:

Total content of flavonoids in NXQ = quercetin content in NXQ × 2.55 + kaempferol content in NXQ × 2.59.

Animal tests

All animal procedures conformed to NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, 1985).

MCAO animal preparation

Adult male Sprague-Dawley (SD) rats (weighing 280 - 320 g, supplied by Guangzhou Medicinal Animal Center, Guangzhou, China) were used for all of the experiments. The animals were kept in a controlled environment at room temperature (RT) of 25 ± 1.0 °C with relative humidity of 40 - 60 %, and a 12 h (6 am - 6 pm) light-dark cycle, and had free access to standard lab chow and tap water. Rat cerebral ischemia was produced by the MCAO (middle cerebral artery occlusion) method as described by Bederson et al. [14] and Longa et al [15]. Two hours after the induction of cerebral ischemia, the reperfusion was conducted.

The neurological behavior of the surviving rats was evaluated at 12, 24, 48, and 72 h after the reperfusion, as described [15]. The five-level score standard was used: 0 score, normal, no symptoms of neurological lesion; 1 score: the rat was unable to fully stretch the front paw controlled by the ischemic cerebral hemisphere; 2 score: the rat moved around along the front paw controlled by the ischemic cerebral hemisphere; 3 score: the rat moved upside down along the front paw controlled by the ischemic cerebral hemisphere; 4 score: rat was unable to move spontaneously. Animals that showed convulsions, half-body paralysis during MCAO for 120 min, and after reperfusion at 10 min, along with those that did not fully develop loss of righting reflex during ischemia, were omitted from the study. In the case of sham-treated animals, the carotid arteries were exposed but not occluded.

Four days after the MCAO, the rats were anesthetized and decapitated. The decapitated rat brains were carefully removed, cooled on ice for 30 min and then coronally sectioned into 2 mm thick sections with a tissue slicer for evaluation of infarct volume and histology by staining with 2 % 2,3,5-triphenyltetrazolium chloride (TTC; Sigma; St. Louis, MO, USA) [14]. The sections obtained were scanned with a CCD camera (Carl Zeiss; Jena, Germany), the total infarct area of the sections was quantified with a CD-8000 Image Analysis System and recalculated to yield the percentage volume of the contralateral hemisphere.

The remnant brain tissues were fixed, frozen, sectioned and stained with hematoxylin and eosin (HE), and then examined under an Olympus bx51 photomicroscope. Attention was especially paid to edema, inflammation, infiltration and necrosis in the brain tissues.

To evaluate the effect of NXQ on cerebral ischemia, in a total of 90 rats were divided into six groups with each group constituting 15 rats. The groups were sham-operated controls, vehicle group, NXQ groups, and EGb761® group.

Drugs and chemicals

NXQ, and EGb761® (purchased from Ipsen France & Dr. Willmar Schwabe) were solubilized in 0.1 % NaHCO₃ solution (pH = 7.5). NXQ was administered at 20, 40, 80 mg/kg/day orally, respectively, for 3 days before the MCAO and 4 days after the MCAO. The same times and routes of administration of 0.1 % NaHCO₃ solution were used for the sham-operated and MCAO ischemia vehicle control rats. All other chemicals used in the present study were of analytical quality. All drug solutions were freshly prepared before use.

On the operation day, NXQ and EGB761® were administered 2 h before the operation and 2 h after the MCAO. The doses (20, 40, 80 mg/kg/day) of NXQ were selected based on the clinical usage, 5 - 7.5 mg/kg/day (p. o.,100 - 150 mg/time, tid) as reported, which demonstrated therapeutic efficacy to cerebral and cardiovascular diseases [1], [3]. EGb761® treated rats were given EGb761® 80 mg/kg , p. o., respectively.

In vitro test

NXQ, rutin and quercetin (Sigma) were dissolved in 60 % alcohol and diluted with serum-free Dulbecco's modified Eagle's medium (DMEM) before use, respectively. The final alcohol concentration was less than 0.01 %. Glutamate was freshly prepared by dissolving in PBS prior to each experiment.

Protection against glutamate toxicity in primary rat hippocampal neuronal culture

Primary rat hippocampal neuronal cultures were prepared from the cerebral hippocampus of neonatal 24 h-old SD rats as described [16] with minor modifications. Briefly, the hippocampus samples were obtained from decapitated 24-h neonatal rats under sterile conditions and cells were mechanically dissociated by triturating them with a set of small-bore, fire-polished Pasteur pipettes and passed through a 200-mesh stainless steel sieve. The cell suspension was centrifuged at 200 × g for 5 min at 4 °C (Eppendorf, Type 5403; Hamburg, Germany). The pellet was re-suspended in DMEM supplemented with 45 % F12 Medium, 5 % fetal bovine/calf serum (Hyclone Co.; Logan, UT, USA), 5 % pregnant horse serum (Hyclone Co.), 100 U/mL penicillin, 100 μg/mL streptomycin) and then plated on poly-D-lysine (MW 30K-700K, Sigma, 10 μg/mL)-coated 24-well tissue culture plates at a density of 2 × 10⁶ cells/cm³, 400 μL per well. The cultures were incubated at 37 °C in a humid 5 % CO₂-95 % atmosphere for 48 h. After plating for 48 hours, cytosine arabinoside (10 μM) was added to the culture to prevent glial cell growth. The medium was removed and refreshed every 48 h. On day 12 in culture, the cultures were refreshed with growth medium and incubated for 2 h, then challenged by L-glutamate (0, 1, 2, 5, 10, 20 μM) by incubation for 10 h in the designated wells. Control cultures were treated with the PBS vehicle at the same time and in the same routine.

In experiments with NXQ, rutin and quercetin, the cells were incubated with 0, 5, 10 and 20 μg/mL of the designated agent 2 h prior to the challenge of glutamate.

Cell viability

After the challenge of glutamate, the cytotoxicity, indicated as neuronal survival viability, was evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma] reduction method [17] _.

Hypoxia test in the primary rat cerebral cortical neuronal culture

Primary rat cerebral cortical neuronal cultures were prepared from the cerebral cortices of neonatal, less than 24-h-old, SD rats as described [18] with minor modifications. Briefly, the whole brains of newborn SD rats (less than 24 h old) were removed and suspended in DMEM. The rat cerebral cortex were then cut into small cubes (1 mm³) and mechanically dissociated by vortexing for 90 s. The cells were then cultured in DMEM containing 10 % fetal bovine serum. The cultures reached confluence at around 2 weeks in vitro and were used in the experiments when they were 12 days old. An anaerobic chamber was used for inducing ischemia [18]. Inside the chamber, the cortical neuronal cultures were washed with the anoxic serum-deprived DMEM three times and then 800 μL/dish of the same medium containing different concentrations of NXQ, rutin and quercetin were added. The cultures were kept in the 37 °C incubator within the anaerobic chamber for 5 h. The atmosphere of the entire unit was saturated with 95 % N₂ - 5 % CO₂ (v/v) and residual oxygen was removed by palladium.

Cell injury was examined by measurement of MTT reduction [17]. Detection of apoptotic cells was determined by staining with the chromatin dye bisbenzimide Hoechst 33 258 (Molecular Probes; Eugene, OR, USA). Cells were fixed for 30 min in the pre-chilled PBS containing 4 % paraformaldehydes with gentle agitation. After fixation, the cells were washed with PBS three times, and then exposed to 2 mg/mL Hoechst 33 258 in the PBS for 10 min at room temperature and washed again three times as before. Cells were examined under a fluorescent microscope (Olympus; Tokyo, Japan). For the determination of the percentage of apoptotic cells, the condensed chromatin nuclei and the total number of nuclei in 12 random fields were determined by microphotography in each culture.

Statistical analysis

All data are expressed as the mean ± SEM and evaluated for statistical significance with the t test or one-way ANOVA followed by Duncan's multiple range test where appropriate. P values < 0.05 were considered to be significant. Our data are drawn from three independent experiments with 10 cultures per experiment in the culture test.

Supporting information

The tables and supplementary figures of the neuroprotective effects of NXQ on the MCAO transient focal cerebral ischemic rats and the cultured neurons injured by glutamate or hypoxia are available as Supporting Information.

Results

There were more than eight major peaks in the fingerprint chromatogram. Protocatechuic acid, quercetin, hyperin, kaempferol in NXQ were identification by LC-MS assays according to their m/z and retention time (Fig. [1], Table 1S and Fig. 1S in the Supporting Information).

The range values for the calibration of quercetin was 2.0 - 30.6 μg/mL (R² = 0.9981), kaempferol 2.0 - 30.6 μg/mL (R² = 0.9984).

Quercetin and kaempferol were identified and quantified in the HCl-hydrolyzed solution. More than 25 % of flavonoids, including about 3.6 % quercetin and its glycosides as well as about 6.3 % kaempferol and its glycosides and some of phenolic acids in NXQ were identified by HPLC assays [19].

The 2-h MCAO transient focal cerebral ischemia followed by reperfusion produced a cerebral hemisphere infarct volume of 79.0 ± 7.0 % in the rats as measured by TTC staining and produced a severe motor incoordination in the rats noted after 12 to 72 h of ischemia (Fig. [2], Table 2S). An increase in the neurological behavior score, an indicator of impairment of neurogical behavior performance was noted in the MCAO-treated rats (Fig. [3], Table 3S).

The administration of NXQ (40, 80 mg/kg/day for 7 days, 3 days before ischemia) markedly reduced the I/R-induced cerebral infarct volume (Fig. [2], Table 2S) and attenuated the increase in neurological behavior score, and improved the I/R-induced motor incoordination observed 12 h to 72 h after ischemia in a dose-dependent fashion (Fig. [3], Table 3S). Administration of EGb761® (80 mg/kg/day) similarly reduced infarct volume by 45.0 ± 17.0 % (P < 0.01; Fig. [2], Table 2S). This suggested that the administration of NXQ evidently inhibited the I/R reaction which lead to an infarct after the 2 h transient cerebral ischemia and improve the neurological behavior of the cerebral I/R rats.

The samples for H&E staining were prepared from the sections adjacent to those for TTC staining. H&E staining showed that the MCAO caused progressive histological damage in rat brains, including disappearance of integrity of the hippocampus, cerebral cortex, and striatum. Marked vacuolation, edema, severe degeneration, infiltration with inflammatory lymphocytes and histological necrosis were also seen in the hippocampuses, cerebral cortexes, and striatum of the ischemic rat brains. The administration of 40, 80 mg/kg NXQ improved the histological alternation in ischemic rat brains with less marked vacuolation, less severe degeneration and less infiltration of lymphocytes (Figs. 2S and 3S in the Supporting Information).

MTT assays showed that the neuronal viability was markedly decreased in a dose-dependent fashion after the cultured hippocampal neurons had been exposed to 2 - 20 μM glutamate. When the glutamate concentration was 2.0, 5.0, 10.0, 20 μM, the neuron viability (mean ± SEM) expressed as percentage of the untreated control was 89.2 ± 9.4 (P < 0.05), 62.8 ± 9.9, 48.5 ± 7.8 and 36.3 ± 7.3 (P < 0.001, n = 9), respectively, suggesting that the hippocampal neurons were very sensitive to glutamate-induced cell injury. The IC₅₀ was about 7.2 μM. At a concentration of less than 500 μg/mL, NXQ showed some increasing effect on the neuron viability. But when the dosage of NXQ was higher than 1000 μg/mL, the viability of the cultured hippocampal neurons was reduced (Table 5S). The IC₅₀ of NXQ was >1000 μg/mL for reducing the viability of cultured hippocampal neurons, suggesting that NXQ could increase the viability of the cultured hippocampal neurons at less than 500 μg/mL and NXQ was of low toxicity to the cultured rat hippocampal neurons. Therefore, to exclude the possible cell injury by the components of NXQ themselves, the dosage of NXQ used in the present study was less than 100 μg/mL.

The exposure of hippocampal neurons to 5 μM glutamate for 10 h caused a sharp decrease of the viability of neurons (p < 0.01). But pre-incubation with 5 - 10 mg/mL NXQ for 2 h prior to the exposure of the hippocampal neurons to 5 μM glutamate could attenuate the decrease of the viability of the hippocampal neurons and even maintain a higher viability of hippocampal neurons in a dose-dependent manner (p < 0.01). Rutin and quercetin produced similar effects, suggesting that NXQ, rutin and quercetin could protect the cultured hippocampal neurons from glutamate- induced cytotoxicity (Table 4S, Fig. [4]).

Our results further demonstrated that preincubation with 5 - 20 μg/mL of NXQ could attenuate glutamate-induced neurotoxicity in a dose-dependent manner. But the effective neuronal protection of rutin and quercetin from glutamate-induced toxicity in the hippocampal neuronal cultures was only observed with concentrations about ≤ 10 μg/mL (Fig. [4]). Rutin and quercetin doses equal to or higher than 20 μg/ml showed little protection (Table 4S, Fig. [4]).

The exposure of the cultured cortical neurons to hypoxia and serum deprivation caused a sharp decrease in the viability of neurons and a marked increase of neuronal apoptosis (p < 0.01, Fig. [5] and Fig. [6], Table 6S and Fig. 4S in the Supporting Information). Preincubation with 5 - 100 mg/mL NXQ for 24 h prior to the exposure of the cortical neurons to hypoxia and serum deprivation could markedly attenuate the decrease of the viability of the cortical neurons in a dose-dependent manner (p < 0.01, Fig. [5], Table 6S).

Approximately 31 % of the cortical neurons died due to apoptosis after their exposure to hypoxia and serum-deprivation. Preincubation of the neurons with 25 - 100 μg/mL NXQ for 2 h conspicuously reduced the neuron apoptotic rate in a dose-dependent manner. At 100 μg/mL NXQ reduced the neuron apoptotic rate to 10.5 ± 2.9 % (P < 0.01, Fig. [6] and Table 6S, Fig. 4S in Supporting Information). This suggested that NXQ could protect the cultured cortical neurons from hypoxia and serum deprivation injury and apoptosis. But 5 - 100 mg/mL rutin and quercetin did not produce similar protective effects (Fig. [5] and Fig. [6], Table 6S and Fig. 4S in Supporting Information).

Discussion

This is the first study to show that administration of NXQ (40, 80 mg/kg, i. g.) markedly protects MCAO rats from focal cerebral I/R injury as demonstrated by the reduction of the infarct volume and the edema area, by the decease in the inflammatory infiltration of leukocytes and necrosis in cerebral tissues and by the improvement in neurological behavior score in rats in a dose-dependent fashion. It is also first to show that NXQ protects primary cultured neurons from excitotoxic glutamate injury and hypoxia injury.

A previous study has demonstrated that the administration of NXQ (40, 80 mg/kg/day, p. o. for 12 days, respectively) increased the survival cell density of hippocampus CA1 pyramidal neurons after transient forebrain ischemia by 4-vessel occlusion in rats [20]. This confirmed that NXQ had produced evident neuroprotection from I/R injury in the MCAO and 4-VO rats.

Free radicals are involved in cerebral ischemia and reperfusion-induced neuronal injury [11]. Cerebral ischemia and reperfusion could cause glutamate release and radicals burst generation, followed by oxidative stress injury [9]. GSH, GSH-Px, catalase, and SOD, along with GSH and other non-enzymatic antioxidants constitute the defence mechanism of cells to protect themselves from damage due to ROS [12]. It was reported that flavonoids and phenolic components containing phenolic hydroxy groups showed a highly potent antioxidant effect, and that they can scavenge ROS, inhibited the formation of ROS, and alleviate the oxidative damage in neuronal cells [21], [22], [23].

We have recently found that pre-incubation with 2 - 30 μg/mL of NXQ alleviated the neuronal injury and apoptosis caused by H₂O₂-induced oxidative-stress in NG108-15 cells in a concentration-dependent fashion by up-regulating bcl-2 expression and by improving the redox equilibrium, as indicated by maintaining the activity of intracellular antioxidant enzymes: CAT, GSH-PX and the content of the intracellular antioxidant GSH, and by reducing the elevation of MDA in the cells [7], [8]. It was also reported that the purified flavonoids and EGb761® protect the neurons by attenuating the depletion of intracellular GSH in the cultured cortical neurons under glutamate toxicity [24], [25]. LC-MS analysis showed that NXQ mainly contains flavonoids including quercetin and its glycosides, hyperin as well as kaempferol and its glycosides, and some phenolic acids like protocatechuic acid. It was also reported that there were benzoic acid, salicylic acid, syringic acid, and scopoletin in NXQ [2], [3]. The phenolic components of NXQ might contribute to the neuroprotection of NXQ against I/R injury in rats and from oxidative stress injury induced by excitotoxic glutamate and hypoxia in the cultured neurons.

It was also reported that in the primary culture of cortical neurons hypoxia and serum deprivation may lead to neuronal damage and apoptosis related to excitory glutamate toxicity, intracellular calcium increase and the formation of superoxide anion radicals. This model mimics physiological ischemia, through severe hypoxia, substrate deprivation and accumulation of toxic metabolites [16], [18].

In the present study, 5 - 100 μg/mL NXQ could improve cell viability and inhibit neuronal apoptosis under the challenge of hypoxia and serum deprivation in the culture of cortical neurons in a dose-dependent fashion (P < 0.05). These findings suggest that NXQ could protect cortical neurons from the injury induced by hypoxia and serum deprivation in the cultures. As to the neuroprotection of NXQ against hypoxia and serum deprivation, non-flavonoid components might also play a more effective role or have a synergistic effect with flavonoids in neuroprotection.

It was also reported that the highest effective concentration of the natural flavonoids, quercetin and kaempfetol, was 100 μM for inhibition of the formation and release of oxide [26]. The IC₅₀ of quercetin for inhibiting the oxidative stress injury induced by H₂O₂ or xanthine (X)/xanthine oxidase (XO) in neurons was about 4 - 5 μg/mL; but no protection was shown when the concentration was equal to or higher than 30 μg/mL [27]. These observations were consistent with our results. It was proposed that the addition of reducing agents (e. g., polyphenolic compounds and quercetin, etc) to commonly used cell-culture media could lead to the generation of substantial amounts of H₂O₂ by interaction of these compounds with cell culture media, which may lead to neuronal apoptosis, and worsen the H₂O₂ formation under hypoxia [28].

Bcl-2 is a key anti-apoptosis mitochondrial protein involved in delayed neuronal death and cerebral ischemic injury. Bcl-2 protects neurons from apoptotic stimuli including oxidative stress and from cerebral ischemic damage [29], [30]. This might suggest that NXQ protects neurons against cerebral ischemic injury, the toxicity of excitatory glutamate and hypoxia injury by up-regulating the expression of bcl-2.

In conclusion, our study confirmed that cultured hippocampal neurons treated with glutamate or cultured cortical neurons challenged by hypoxia caused neuronal apoptosis with a marked decrease in cell viability, and that NXQ could protect cerebral tissues from I/R injury, reduce the cerebral infarct volume and the cerebral edema and improve the neurological behavior of rats suffering from MCAO. NXQ could also attenuate the I/R damage of neurons, inhibit neuronal apoptosis and neuronal necrosis, and potently protect the neurons from excitotoxical glutamate-induced injury and hypoxia-induced damage. These observations might represent the mechanism underlying NXQ's potent efficacy in the prevention and treatment of stroke or apoplexy syndrome, which may be much more complex and involve synergy between the effects of different components, such as the flavonoids and the other polyphenol components.

Acknowledgments

This work was supported by grants from the Guangzhou Commission of Science and Technology, PR China (2002Z2-E5081). The authors are most grateful to Dr. Jianhui Zhong, Dr. Zhan Zhou, Dr. Wen Huang, Dr Ailin Liu and Ms. Songmin He for their excellent technical aid in the MCAO model, Mr Jie Luo for HPLC assay help, Mr Junhua Yao for LC-MS assays help. The paper was proof-read by Professor Yidun Guo of the English Teaching Department, School of Foreign Language, Sun Yat-sen University

References

• 1 Cai Y D, Yang S F. Effect of Naoxinqing tablet for cerebral atherosclerosis and angina pectoris of coronary heart disease: an observation of 60 cases.  Trad Chin Drug Res Clin Pharmacol. 2001;  12 414-6.
  PubMedSearch in Google Scholar
• 2 Huang S L, Hu X G, Chen D C, Xiao L. Effects of Naoxinning on cerebral blood flow in anesthetized dogs.  Guangxi Med J. 1987;  9 124-5.
  PubMedSearch in Google Scholar
• 3 Yu Y Z, Yu Z Y, Guo J. The experimental and clinical studies of Naoxinning in the treatment of ischemic cerebral vascular disease.  Med J Chin People Liberation Army. 1988;  13 30-1.
  PubMedSearch in Google Scholar
• 4 Huang S L, Lin X L, Chen L F. Effects of extract of leaves of Diospyros kaki on heart Function and hemodynamics in anathesized dogs.  Chin Pharm J. 1983;  18 372.
  PubMedSearch in Google Scholar
• 5 Liang C, Fu F M, Zhang K S. Cardiovascular effects of Diospyros kaki leaves.  Chin Pharm J. 1985;  20 245-6.
  PubMedSearch in Google Scholar
• 6 Huang S L, Nong X X, Li Y T, Chen D C, Wu Y Q, YIN Y Q. Effects of the extract of leaves of Diospyros kaki on hemorrheology in rabbits.  Chin Pharm J. 1983;  18 372.
  PubMedSearch in Google Scholar
• 7 Bei W J, Peng W L, Ma Y, Xu A L. Naoxinqing, an anti-stroke herbal medicine, reduces hydrogen peroxide-induced injury in NG108 - 15 cells.  Neurosci Lett. 2004;  363 262-5.
  CrossrefPubMedSearch in Google Scholar
• 8 Bei W J, Peng W L, Ma Y, Xu A L. Flavanoids from leaves of Diospyros kaki reduces hydrogen peroxide-induced injury in NG108-15 cells.  Life Sci. 2005;  76 1975-88.
  CrossrefPubMedSearch in Google Scholar
• 9 Coyle J T, Puttfarcken P. Oxidative stress, glutamate and neurodegenerative disorder.  Science. 1993;  262 689-95.
  CrossrefPubMedSearch in Google Scholar
• 10 Globus M Y-T, Alonsa O, Dietrich W D, Busto R, Ginsberg M D. Glutamate release and free radical production following brain injury: effects of posttraumatic hypothermia.  J Neurochem. 1995;  65 1704-11.
  PubMedSearch in Google Scholar
• 11 Facchinetti F, Dawson V L, Dawson T M. Free radicals as mediator of neuronal injury.  Cell Mol Neurobiol. 1988;  18 667-82.
  PubMedSearch in Google Scholar
• 12 Yassi G -S, Ziv R, Eldad M, Daniel O. Antioxidant therapy in acute central nervous system injury: current state.  Pharmacol Rev. 2002;  54 271-84.
  CrossrefPubMedSearch in Google Scholar
• 13 Chandrasekaran K, Mehrabian Z, Spinnewyn B, Chinopoulos C, Drieu K, Fiskum G. Neuroprotective effects of Bilobalide, a component of Ginkgo biloba extract (EGb761®) in global brain ischemia and in excitotoxicity-induced neuronal death.  Pharmacopsychaiatry. 2003;  36 S89-94.
  PubMedSearch in Google Scholar
• 14 Bederson J B, Pitts L H, Tsuji M, Germano S M, Nishimura M C, Davis R L. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats.  Stroke. 1986;  17 1304-8.
  PubMedSearch in Google Scholar
• 15 Longa E Z, Weinstein P R, Carlson S. Reversible middle cerebral artery occlusion without craniectomy in rats.  Stroke. 1989;  20 84-91.
  CrossrefPubMedSearch in Google Scholar
• 16 Oillet J, Koziel V, Vert P, DavL J L. Influence of post-hypoxia reoxygenation conditions on energy metabolism and superoxide production in cultured neurons from the rat forebrain.  Pediatr Res. 1996;  39 598-603.
  CrossrefPubMedSearch in Google Scholar
• 17 Hansen M B, Nielsen S E, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.  J Immunol Methods. 1989;  119 203-10.
  CrossrefPubMedSearch in Google Scholar
• 18 Goldberg M P, Choi D W. Combined oxygen and glucose deprivation in cortical cell culture: Calicium-dependent and Calicium-independent mechanisms of neuronal injury.  J Neurosci. 1993;  13 3510-24.
  PubMedSearch in Google Scholar
• 19 Bei W J, Luo J, Peng W L, Wu A L. The Determination of flavonoids in the extract of the leaves of Diospyros kaki by HPLC.  Chin Tradit Herbal Drugs. 2005;  36 014-5.
  PubMedSearch in Google Scholar
• 20 Bei W J, Zhu X H, Peng W L. Neuroprotection of Naoxingqing tablet against neuronal injury in hippocampus, after transient forebrain ischemia in rat.  Practice Chin Clin Med. 2005;  2 13-6.
  PubMedSearch in Google Scholar
• 21 Ishige K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms.  Free Radic Biol Med. 2001;  30 433-46.
  CrossrefPubMedSearch in Google Scholar
• 22 Robak J, Gryglewski R J. Flavonoids are scavengers of superoxide anions.  Biochem Pharmacol. 1998;  37 837-41.
  PubMedSearch in Google Scholar
• 23 Afanas'ev I B, Dorozhko A I, Brodskii A V, Kostyuk V A, Potapowitch A I. Chelating and free radical scavenging mechanisms of inhibitory action of rutrin and quercentin in lipid peroxidation.  Biochem Pharmacol. 1989;  38 1763-9.
  CrossrefPubMedSearch in Google Scholar
• 24 Almeida A. Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion.  Brain Res. 1998;  790 209-16.
  CrossrefPubMedSearch in Google Scholar
• 25 Kim S R, Park M J, Lee M K, Sung S H, Park E J, Kim J. Flavonoids of Inula britannica protect cultured cortical cells from necrotic cell death induced by glutamate.  Free Radic Biol Med. 2002;  32 596 -604.
  CrossrefPubMedSearch in Google Scholar
• 26 Zielinska M, Kostrzewa A, Ignatowicz E. Antioxidative activity of flavonoids in stimulated human neutrophils.  Folia Histochem Cytobiol. 2000;  38 25-30.
  PubMedSearch in Google Scholar
• 27 Nagao A, Seki M, Kobayashi H. Inhibition of xanthine oxidase by flavonoids.  Biosci Biotechnol Biochem. 1999;  63 1787-90.
  PubMedSearch in Google Scholar
• 28 Halliwell B, Clement M V, Ramalingam J, Long L H. Hydrogen peroxide. Ubiquitous in cell culture and in vivo?.  IUBMB Life. 2000;  50 251-7.
  CrossrefPubMedSearch in Google Scholar
• 29 Allsopp T E, Wyatt S, Paterson M F, Davies A M. The protooncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis.  Cell. 1993;  73 295-307.
  CrossrefPubMedSearch in Google Scholar
• 30 Martinou J C, Dubois-Dauphin M, Staple J K. Overexpression of Bcl-2 in transgene mice protects neurons from naturally occurring cell death and experimental ischemia.  Neuron. 1994;  13 1017-30.
  CrossrefPubMedSearch in Google Scholar

Prof. Anlong Xu

Department of Biochemistry

School of Life Sciences

Sun Yat-sen University

135 W Xingang Road

Guangzhou 510275

Guangdong

People's Republic of R China

Phone: +86-20-8411-3655

Fax: +86-20-8403-8377

Email: lssxal@mail.sysu.edu.cn

References

• 1 Cai Y D, Yang S F. Effect of Naoxinqing tablet for cerebral atherosclerosis and angina pectoris of coronary heart disease: an observation of 60 cases.  Trad Chin Drug Res Clin Pharmacol. 2001;  12 414-6.
  PubMedSearch in Google Scholar
• 2 Huang S L, Hu X G, Chen D C, Xiao L. Effects of Naoxinning on cerebral blood flow in anesthetized dogs.  Guangxi Med J. 1987;  9 124-5.
  PubMedSearch in Google Scholar
• 3 Yu Y Z, Yu Z Y, Guo J. The experimental and clinical studies of Naoxinning in the treatment of ischemic cerebral vascular disease.  Med J Chin People Liberation Army. 1988;  13 30-1.
  PubMedSearch in Google Scholar
• 4 Huang S L, Lin X L, Chen L F. Effects of extract of leaves of Diospyros kaki on heart Function and hemodynamics in anathesized dogs.  Chin Pharm J. 1983;  18 372.
  PubMedSearch in Google Scholar
• 5 Liang C, Fu F M, Zhang K S. Cardiovascular effects of Diospyros kaki leaves.  Chin Pharm J. 1985;  20 245-6.
  PubMedSearch in Google Scholar
• 6 Huang S L, Nong X X, Li Y T, Chen D C, Wu Y Q, YIN Y Q. Effects of the extract of leaves of Diospyros kaki on hemorrheology in rabbits.  Chin Pharm J. 1983;  18 372.
  PubMedSearch in Google Scholar
• 7 Bei W J, Peng W L, Ma Y, Xu A L. Naoxinqing, an anti-stroke herbal medicine, reduces hydrogen peroxide-induced injury in NG108 - 15 cells.  Neurosci Lett. 2004;  363 262-5.
  CrossrefPubMedSearch in Google Scholar
• 8 Bei W J, Peng W L, Ma Y, Xu A L. Flavanoids from leaves of Diospyros kaki reduces hydrogen peroxide-induced injury in NG108-15 cells.  Life Sci. 2005;  76 1975-88.
  CrossrefPubMedSearch in Google Scholar
• 9 Coyle J T, Puttfarcken P. Oxidative stress, glutamate and neurodegenerative disorder.  Science. 1993;  262 689-95.
  CrossrefPubMedSearch in Google Scholar
• 10 Globus M Y-T, Alonsa O, Dietrich W D, Busto R, Ginsberg M D. Glutamate release and free radical production following brain injury: effects of posttraumatic hypothermia.  J Neurochem. 1995;  65 1704-11.
  PubMedSearch in Google Scholar
• 11 Facchinetti F, Dawson V L, Dawson T M. Free radicals as mediator of neuronal injury.  Cell Mol Neurobiol. 1988;  18 667-82.
  PubMedSearch in Google Scholar
• 12 Yassi G -S, Ziv R, Eldad M, Daniel O. Antioxidant therapy in acute central nervous system injury: current state.  Pharmacol Rev. 2002;  54 271-84.
  CrossrefPubMedSearch in Google Scholar
• 13 Chandrasekaran K, Mehrabian Z, Spinnewyn B, Chinopoulos C, Drieu K, Fiskum G. Neuroprotective effects of Bilobalide, a component of Ginkgo biloba extract (EGb761®) in global brain ischemia and in excitotoxicity-induced neuronal death.  Pharmacopsychaiatry. 2003;  36 S89-94.
  PubMedSearch in Google Scholar
• 14 Bederson J B, Pitts L H, Tsuji M, Germano S M, Nishimura M C, Davis R L. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats.  Stroke. 1986;  17 1304-8.
  PubMedSearch in Google Scholar
• 15 Longa E Z, Weinstein P R, Carlson S. Reversible middle cerebral artery occlusion without craniectomy in rats.  Stroke. 1989;  20 84-91.
  CrossrefPubMedSearch in Google Scholar
• 16 Oillet J, Koziel V, Vert P, DavL J L. Influence of post-hypoxia reoxygenation conditions on energy metabolism and superoxide production in cultured neurons from the rat forebrain.  Pediatr Res. 1996;  39 598-603.
  CrossrefPubMedSearch in Google Scholar
• 17 Hansen M B, Nielsen S E, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.  J Immunol Methods. 1989;  119 203-10.
  CrossrefPubMedSearch in Google Scholar
• 18 Goldberg M P, Choi D W. Combined oxygen and glucose deprivation in cortical cell culture: Calicium-dependent and Calicium-independent mechanisms of neuronal injury.  J Neurosci. 1993;  13 3510-24.
  PubMedSearch in Google Scholar
• 19 Bei W J, Luo J, Peng W L, Wu A L. The Determination of flavonoids in the extract of the leaves of Diospyros kaki by HPLC.  Chin Tradit Herbal Drugs. 2005;  36 014-5.
  PubMedSearch in Google Scholar
• 20 Bei W J, Zhu X H, Peng W L. Neuroprotection of Naoxingqing tablet against neuronal injury in hippocampus, after transient forebrain ischemia in rat.  Practice Chin Clin Med. 2005;  2 13-6.
  PubMedSearch in Google Scholar
• 21 Ishige K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms.  Free Radic Biol Med. 2001;  30 433-46.
  CrossrefPubMedSearch in Google Scholar
• 22 Robak J, Gryglewski R J. Flavonoids are scavengers of superoxide anions.  Biochem Pharmacol. 1998;  37 837-41.
  PubMedSearch in Google Scholar
• 23 Afanas'ev I B, Dorozhko A I, Brodskii A V, Kostyuk V A, Potapowitch A I. Chelating and free radical scavenging mechanisms of inhibitory action of rutrin and quercentin in lipid peroxidation.  Biochem Pharmacol. 1989;  38 1763-9.
  CrossrefPubMedSearch in Google Scholar
• 24 Almeida A. Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion.  Brain Res. 1998;  790 209-16.
  CrossrefPubMedSearch in Google Scholar
• 25 Kim S R, Park M J, Lee M K, Sung S H, Park E J, Kim J. Flavonoids of Inula britannica protect cultured cortical cells from necrotic cell death induced by glutamate.  Free Radic Biol Med. 2002;  32 596 -604.
  CrossrefPubMedSearch in Google Scholar
• 26 Zielinska M, Kostrzewa A, Ignatowicz E. Antioxidative activity of flavonoids in stimulated human neutrophils.  Folia Histochem Cytobiol. 2000;  38 25-30.
  PubMedSearch in Google Scholar
• 27 Nagao A, Seki M, Kobayashi H. Inhibition of xanthine oxidase by flavonoids.  Biosci Biotechnol Biochem. 1999;  63 1787-90.
  PubMedSearch in Google Scholar
• 28 Halliwell B, Clement M V, Ramalingam J, Long L H. Hydrogen peroxide. Ubiquitous in cell culture and in vivo?.  IUBMB Life. 2000;  50 251-7.
  CrossrefPubMedSearch in Google Scholar
• 29 Allsopp T E, Wyatt S, Paterson M F, Davies A M. The protooncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis.  Cell. 1993;  73 295-307.
  CrossrefPubMedSearch in Google Scholar
• 30 Martinou J C, Dubois-Dauphin M, Staple J K. Overexpression of Bcl-2 in transgene mice protects neurons from naturally occurring cell death and experimental ischemia.  Neuron. 1994;  13 1017-30.
  CrossrefPubMedSearch in Google Scholar

Prof. Anlong Xu

Department of Biochemistry

School of Life Sciences

Sun Yat-sen University

135 W Xingang Road

Guangzhou 510275

Guangdong

People's Republic of R China

Phone: +86-20-8411-3655

Fax: +86-20-8403-8377

Email: lssxal@mail.sysu.edu.cn

• Supplementary Material