Neuroscience Letters 363 (2004) 262–265

www.elsevier.com/locate/neulet

NaoXinQing, an anti-stroke herbal medicine, reduces hydrogen peroxide-

induced injury in NG108-15 cells
WeiJian Beia,b, Wenlie Penga, Yan Maa, Anlong Xua,*
a
Department of Biochemistry, College of Life Sciences, Sun Yatsen (Zhongshan) University, 135 W. Xingang Road, Guangzhou, Guangdong, 510275,
PR China
b
Guangzhou Pharmaceutical Holdings Limit, BaiYunShan TCM Pharmaceutical Factory, 389 N. Shatai Road, Guangzhou, Guangdong, 510515, PR China
Received 3 March 2004; received in revised form 31 March 2004; accepted 1 April 2004

Abstract
NaoXinQing (NXQ) is a patented and approved drug of Traditional Chinese Medicine that has been used for years for the treatment of
syndrome of apoplexy, but the underlying mechanism remains unclear. The present study is designed to investigate the effects of NXQ on
hydrogen peroxide (H2O2)-induced cell damage in NG108-15 cells. Exposure to H2O2 induces apoptosis-like cell injury. Preincubation of
cells with NXQ alleviated H2O2-induced cell injury and apoptosis. This herb medicine also improves redox imbalance in cells under the
exposure of H2O2 as indicated by the attenuation in the reduction of activities of intracellular endogenous antioxidants, glutathione and
glutathione peroxidase as well as catalase, and by the decrease in the leak of lactate dehydrogenase and the accumulation of
malondialdehyde. These results indicate that NXQ significantly protects NG108-15 cells against H2O2 challenge by improving redox
imbalance and inhibiting apoptosis, which might represent mechanisms underlying its potential usage in the prevention and treatment of
syndrome of apoplexy.
q 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: NaoXinQing; Diospyros kaki L.; Antioxidant; Oxidative stress; Apoptosis; NG108-15 cell; H2O2

NaoXinQing (NXQ) is a patented and approved drug of increase electrophoretic mobility of erythrocyte, lower the
Traditional Chinese Medicine (TCM) made from the extract blood or plasma viscosity and decrease the deposit of
of leaves of Diospyros kaki L. Dispryosl and Ebenceae, and fibrinogen in rabbit [9,11]. Despite these clinical obser-
has been used for years for the treatment of stroke or vations and pharmacological research, the precise mechan-
syndrome of apoplexy in clinics in China to improve the isms for the treatment of this drug in stroke remain unclear.
outcome of ischemia stroke [3]. The remedy has been The reactive oxygen species (ROS), mainly including
reported to possess superior efficacy and very few side- superoxide anion radical (zO2 2 ), hydrogen peroxide (H2O2),
effects in the treatment of stroke patients such as cerebral and hydroxyl radical (OHz), are highly reactive molecules
atherosclerosis, transitory ischemia syndrome (TIS), cer- generated predominantly during cellular respiration and
ebral thrombogenesis, cerebral thrombosis sequela, apo- normal metabolism by different oxidases, oxygenases, or by
plexy sequela, cerebral embolism, etc. [3,9,17]. auto-oxidation of catecholamines. Defense mechanisms of
Previous studies have demonstrated that the extract from cells against oxidative damage are involved in enzymatic
the leaves of D. kaki L. could increase coronary blood flow conversion of ROS into less reactive molecules, chelation of
and reduce its resistance in anesthetized dog, antagonize the transition metal catalysts and detoxification of ROS by
main artery constriction by potassium chloride in isolated antioxidants. Continuous and excessive production of ROS,
rabbit aortic vessels, improve the cardiac function, lower the which is referred to as oxidative stress, will inevitably lead
myocardiac oxygen consumption, and improve the overall
to exposure of cells to ROS directly, which subsequently
circulation [9,11,17]. Besides, it could also significantly
react with and damage cellular components such as proteins,
* Corresponding author. Tel.: þ 86-20-84113655; fax: þ 86-20-
lipids, and DNA, as well as generating other toxic products
84038377. from these reactions. ROS finally can lead to subsequent cell
E-mail address: ls36@zsu.edu.cn (A. Xu). death by mode of necrosis and apoptosis [2,15,16].
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2004.04.031
 W.J. Bei et al. / Neuroscience Letters 363 (2004) 262–265 263

Oxidative stress is generally known to be involved in acute incubated at 37 8C for 30 min, and then with PI 50 mg/ml
and chronic central nervous system (CNS) injury and is a and 0.1% NP40 in the dark for 30 min. The cell suspension
major factor in determining the development of neuronal was assayed by FCM (Beckton-Dickinson FACSCALI-
damage [1,5]. The brain is especially susceptible to the BUR) after removing cellular debris by filtration.
damage caused by oxidative stress because neurons contain To assess the antioxidants and lipid peroxide in cells after
enriched polyunsaturated fatty acids and low levels of H2O2 insult, the cultures were harvested and washed with
endogenous antioxidant enzymes [5]. Acute ischemic strokes ice-cold PBS and then pooled in 0.1 M PBS/0.05 mM
can cause neuron apoptosis by oxidative stress, which might EDTA buffered solution and homogenized. The homogen-
be mainly induced by a burst of the generation of ROS during ate was centrifuged for 1 h at 10,000 £ g at 4 8C. The
cerebral ischaemia, especially at the onset of reperfusion after supernatants were used in the assay [15]. We assessed the
cerebral ischaemia [1,2,16]. Some treatment with antioxidants content of protein in cells by the Coomassie blue protein-
aimed at scavenging free radicals or preventing their binding method with bovine serum albumin as standard.
formation therefore might be a reasonable selection for stroke Glutathione (GSH) content was assayed with a GSH assay
and other neurodegenerative diseases [12,15–16]. kit and the content of malondialdehyde (MDA) was
H2O2, a major ingredient of ROS, has been extensively determined at 532 nm with an MDA assay kit on the basis
used to induce oxidative stress in in vitro models [14,15]. of a modified thiobarbituric acid method. Glutathione
We carried out the present study to examine the effects of peroxidase (GSH-Px) was measured at 412 nm with a
NXQ on H2O2-induced injury of NG108-15 cells. GSH-Px assay kit by quantifying the rate of oxidation of
NXQ, supplied by BaiYunShan TCM Pharmaceutical reduced GSH to oxidized glutathione (GS-GS). Catalase
Factory, was the extract of leaves of D. kaki L. The main activity was analyzed at 405 nm with a catalase assay kit
composition of NXQ is more than 50% organic acids based on the H2O2 decomposition. Assay kits for catalase,
(mainly including protocatechuic acid, benzoic acid, GSH, GSH-Px and MDA were all from Nanjing Jian Chen
salicylic acid, syringic acid, pyromucic acid, succinic acid BioChem Co.
and scopoletin, etc.) [18] and 26.56% flavonoids, including Results were drawn from two to three independent
quercetin and its glucosides (hyperin, isoquercitrin) as well experiments with five to ten cultures per experiment, except
as kaempfetol and its glucosides (astragalin), etc. (data not for the flow cytometric assay. All results are shown as the
shown). mean ^ SEM, and statistical significance was evaluated by
NXQ was dissolved in an aqueous solution of 10% t-test or one-way analysis of variance (ANOVA) followed
DMSO with double distilled water and diluted with serum- by Duncan’s multiple range test where appropriate.
free RPMI 1640 medium (Gibco) before use. P , 0:05 was regarded as statistically significant.
The NG108-15 cell was obtained from the Shanghai Cell MTT assays showed that cell viability was strikingly
Institute, Chinese Academy of Science. Cell culture was reduced in a dose-dependent fashion after NG108-15 cell
performed as described by Hamprecht [6]. Cells were plated cultures were exposed to 200 – 1000 mM H2O2, confirming
into multi-well plates or dishes (Falcon Co.) at a density of that NG108-15 cells were particularly susceptible to H2O2-
5 –8 £ 104 cells/ml in RPMI 1640 medium (Gibco) contain- induced cell damage [14]. The IC50 of H2O2 in NG108-15
ing 10% fetal bovine serum (Hyclone Co.), 100 units/ml of cells was 420.5 mM. At a concentration of less than 100 mg/
ampicillin, and 100 mg/ml of streptomycin, and cultured at ml, NXQ showed little effect on cell viability, which was
37 8C in a damped gas mixture of 5% CO2/95% air. Cells markedly decreased in a dose-dependent manner after NXQ
were tested at the log-phase, about 48 h after seeding. We concentrations higher than 500 mg/ml. When the NXQ
freshly prepared H2O2 from 30% stock solution (Sigma) concentration was 100, 500, 1000, and 1500 mg/ml, the cell
before each test. The cytotoxicity of NXQ and H2O2, an viability (mean ^ SEM expressed as a percentage of the
indication of cell viability, was measured by the MTT (3- non-H2O2-treated control, n ¼ 7 – 10) was 98.3 ^ 10.1
(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bro- (P . 0:05), 76.3 ^ 29.6 (P , 0:05), 49.8 ^ 15.7
mide, Sigma) reduction method [8,15]. Lactate dehydro- (P , 0:001), and 27.3 ^ 7.0 (P , 0:001), respectively.
genase (LDH) leakage in extracellular supernatant medium, Therefore, to exclude the possible cell injury by NXQ
an index of cell injury, was tested with an LDH assay kit at itself, the dosage of NXQ used in the present study was less
440 nm (Nanjing Jian Chen BioChem Co.). than 100 mg/ml. Our results further demonstrated that
Detection of apoptotic cells was determined with preincubation with 2 –30 mg/ml of NXQ could alleviate
chromatin dye Hoechst 33258 (Molecular Probe) staining H2O2-induced cell injury. The maximum protection of NXQ
as described by Han [7]. We determined the percentage of from H2O2-induced NG108-15 cell injury was observed at
apoptotic cells by flow cytometry (FCM) as described by the concentration of 30 mg/ml (data shown in Table 1).
Darzynkiewcz [4] with some modifications. After being Our study showed that LDH leakage in extracellular
induced by H2O2, cells were harvested by centrifugation and culture medium was drastically increased by about 88%
washed with PBS. In each sample, about 10 million cells after NG108-15 cells were exposed to 300 mM H2O2 for 10
were collected and fixed with 70% ethanol at 4 8C overnight. h. However, when the cells were preincubated with 2 – 30
The cells were added with RNase A (60 mg/ml), and mg/ml of NXQ, the increase of H2O2-induced LDH leakage
264 W.J. Bei et al. / Neuroscience Letters 363 (2004) 262–265

Table 1
Protection of NXQ from H2O2 injury in NG108-15 cells

Group Cell viability (OD value) LDH leakage (KU/gprot) Cell apoptosis

Control 100 (0.347 ^ 0.027) 100 ^ 7.3 (29.3 ^ 2.1) 3.45 ^ 0.78
NXQ 30 mg/ml 95.4 ^ 2.3## 116.4 ^ 13.2## 12.4 ^ 3.8##
NXQ 10 mg/ml 90.5 ^ 4.3## 148.8 ^ 14.4## 21.5 ^ 1.2##
NXQ 2 mg/ml 91.9 ^ 3.2## 145.8 ^ 12.0## 30.3 ^ 0.8#
H2O2 65.6 ^ 12.4** 188.2 ^ 22.9** 33.1 ^ 1.1**

Cells were challenged with 300 mM H2O2 for 10 h; NXQ was added 2 h before the addition of H2O2. Cell viability was tested by evaluating the MTT
reduction 10 h later (n ¼ 7 – 10), and the activity of LDH in the extracellular medium was assayed using LDH assay kits (n ¼ 5 – 7). The percentage of
apoptotic cells was measured by FCM (n ¼ 3 – 5). The results are the mean ^ SEM of n separate experiments shown as a percentage of the corresponding non-
H2O2-treated control (n ¼ 3 – 10). Statistical significance for comparison was evaluated by ANOVA followed by Dunnett’s test. Two to three independent
experiments were carried out. *P , 0:05, **P , 0:01 vs. non-H2O2-treated control; #P , 0:05, ##P , 0:01 vs. H2O2-treated group.

into the medium was markedly alleviated dose-dependently damage due to ROS [15]. To test if preincubation with NXQ
(Table 1), further indicating that NXQ could alleviate will modulate endogenous antioxidants after NG108-15
NG108-15 cell injury by H2O2. cells are treated with H2O2, GSH content and GSH-Px as
Next, we explored whether NXQ alleviates H2O2- well as catalase activity were assayed.
induced cell injury by decreasing cell apoptosis. As After NG108-15 cells were exposed to H2O2 (300 mM)
shown in Fig. 1 and Table 1, approximately 33% of cells for 10 h, the GSH content and GSH-Px and catalase
died due to apoptosis after exposure to H2O2. Preincu- activities were sharply decreased by about 28%, 48% and
bation of the cells with 30 mg/ml NXQ for 2 h 65%, respectively. By contrast, MDA was drastically
conspicuously reduced the cell apoptotic rate to increased by about 69%. Pretreatment with NXQ signifi-
12.4 ^ 3.8% (P , 0:01) (Table 1), suggesting a protec- cantly reversed the alterations in GSH and MDA levels and
tive effect of NXQ on H2O2-mediated cell apoptosis. GSH-Px and catalase activities (Table 2).
GSH-Px and catalase are the most important endogenous The clinical dosage of NXQ is 5.0– 10.0 mg/kg (100 –
antioxidant enzymes, and along with superoxide dismutase 200 mg/time, tid) [3,17]. We estimate that the NXQ
(SOD) and GSH as well as other non-enzymatic antiox- concentration in the plasma may reach 31.3– 125 mg/ml.
idants constitute the defense mechanism of cells against Supposing that 10– 30% NXQ in the plasma could be
transported into the brain tissue in vivo, the NXQ
concentration in the brain tissue might reach 3.1– 37.5 mg/
ml. Although NXQ itself at a higher concentration (higher
than 100 mg/ml) has cellular toxicity, we can suppose that
the clinical dosage should be an effective but non-cellular
toxic concentration (range 2 –30 mg/ml) in the insulting
brain tissue in vivo.
NXQ mainly contained flavonoids (including quercetin,
kaempfetol and their glucosides, such as astragalin, rutin,
and hyperin) and some phenolic acids, such as protocate-
chuic acid, benzoic acid, salicylic acid and syringic acid
[18]. Some of these flavonoids and phenolic compounds
show very potent antioxidant effects and radical scavenger
effects [10,13], which may be responsible for NXQ’s
protection on H2O2-induced injury in NG108-15 cells.
Collectively, our present studies demonstrated that
NG108-15 cells challenged by H2O2 caused apoptosis
Fig. 1. Protection of NXQ against apoptosis induced by 300 mM H2O2 in with a striking decrease in cell viability and an imbalance of
NG108-15 cells. Cells were exposed to 300 mM H2O2 for 10 h. NXQ was the redox state with an accumulation of MDA, LDH release
added to the culture 2 h before the addition of H2O2. Cells were harvested
and fixed in paraformaldehyde and examined under a LEICA DMIL
into culture medium and a decrease of GSH content and
MPS30-inverted phase-contrast fluorescence microscope ( £ 400) by GSH-Px and catalase activities. Preincubation with NXQ
staining with Hoechst 33258 dye. (A) Non-H2O2-treated control NG108- potently attenuated oxidative stress and protected NG108-
15 cells; (B) NG108-15 cells challenged by 300 mM H2O2; (C) NG108-15 15 cells from injury induced by H2O2, which might
cells challenged by 300 mM H2O2 in the presence of 10 mg /ml NXQ. The
figure represents three experiments with similar results. Hoechst 33258 dye
represent the mechanism underlying their potent usage in
was used to stain the DNA of the shrunken nuclei and the chromatin the prevention and treatment of stroke or syndrome of
condensation in nuclei in the fluorescence pictures. apoplexy.
 W.J. Bei et al. / Neuroscience Letters 363 (2004) 262–265 265

Table 2
Effect of NXQ on the H2O2-induced alteration of CAT, GSH-PX and the content of GSH and MDA in the cytoplasm of NG108-15 cells

Group Catalase (units/mg protein) GSH-Px (units/mg protein) GSH (mg/g protein) MDA (nM/mg protein)

Control 100 ^ 7.9 (4.42 ^ 0.56) 100 ^ 9.8 (68.8 ^ 6.76) 100 ^ 12.5 (123.9 ^ 15.6) 100 ^ 11.2 (1.43 ^ 0.16)
H2O2 34.8 ^ 12.5** 52.2 ^ 8.3** 72.2 ^ 11.28** 169.2 ^ 18.6**
H2O2 þ NXQ 2 mg/ml 52.9 ^ 13.9## 60.2 ^ 8.2## 89.9 ^ 14.0# 147.1 ^ 19.3##
H2O2 þ NXQ 10 mg/ml 57.0 ^ 14.1## 70.7 ^ 6.4## 105.7 ^ 33.5## 130.0 ^ 20.7##
H2O2 þ NXQ 30 mg/ml 93.2 ^ 12.9## 88.9 ^ 11.5## 139.2 ^ 29.9## 115.0 ^ 17.9##

Cells were incubated with 300 mM H2O2 for 10 h. NXQ was added 2 h before the addition of H2O2. Then the activity of catalase and GSH-Px and the
contents of GSH and MDA in the cytoplasm were tested by corresponding kits. The results are the mean ^ SEM shown as percentages of the corresponding
non-H2O2-treated control (n ¼ 5 – 7) in two independent experiments. Statistical significance was evaluated by ANOVA followed by Dunnett’s test.
*P , 0:05, **P , 0:01 vs. non-H2O2-treated control; #P , 0:05, ##P , 0:01 vs. 300 mM H2O2-treated group.

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