558 Biol. Pharm. Bull. 24(5) 558—563 (2001) Vol. 24, No.

Antioxidant Potential of Qizhu Tang, a Chinese Herbal Medicine, and the

Effect on Cerebral Oxidative Damage after Ischemia Reperfusion in Rats
Wang XUEJIANG,1) Haruyo ICHIKAWA, and Tetsuya KONISHI*
Department of Radiochemistry-Biophysics, Niigata College of Pharmacy, 5–13–2 Kamishin-ei, Niigata 950–2081 Japan.
Received August 25, 2000; accepted January 26, 2001

A traditional Chinese herbal medicine, Qizhu Tang (QZT) was studied for its in vitro antioxidant activity
and the effect on cerebral oxidative damage after forebrain ischemia followed by reperfusion in rats. The QZT
decoction was shown to have strong hydroxyl radical (· OH) scavenging activity (approx. 0.1 mM as Trolox equiva-
lent) when determined by ESR using DMPO as a spin trap reagent and H2O2/UV as the · OH source. When the
QZT decoction was injected into rat duodenum 2 h before cerebral ischemia, the oxidative brain damage after
45 min reperfusion was strongly inhibited in terms of two biochemical indications, thiobarbituric acid reactive
substance formation and the loss of glutathione peroxidase. Since the QZT formula consists of 4 herbal con-
stituents (Rhizoma atractylodis, Poria, Radix notoginseng and Radix astragali), each of the component herbs and
their combinations were also examined for their protective effects on the cerebral ischemia/reperfusion injury
and the effects were compared with their in vitro antioxidant potential. Although some of the incomplete formu-
las showed as strong antioxidant activities as complete QZT in vitro, only the complete QZT formula was effective
in preventing cerebral oxidative injury in rats, and other preparations showed limited activity in vivo.
Key words Traditional Chinese medicine; antioxidant herb medicine; cerebral oxidative damage; ischemia-reperfusion; Qizhu
Tang; composite formula

It is now apparent that the oxidative stress induced by re-
active oxygen and nitrogen species is implicated in aging,
carcinogenesis and many other pathophysiological condi-
tions2—5) as a causative factor. Antioxidant therapy thus has
been proposed for the treatment of these disorders.6) How-
ever, the single use of antioxidants is usually less effective
than expected. The therapeutic potential of traditional herbal
medicines however, is attracting attention as an alternative
medicine for treating diseases related to lifestyle.7) Since re-
active oxygen species are implicated in these diseases,8) an-
tioxidative Chinese traditional medicines would be a reliable
target of study for their potential to prevent and repair oxida-
tive stress-related pathological conditions. Usually, the for-
mula for a Chinese herbal medicine consists of several com-
ponents with different functions. Therefore, it seems to be a
good model for an antioxidant-based composite therapy, in
that the combination of several components with different
functions is designed to ameliorate the pathophysiological
conditions related to oxidative stress.
Brain oxidative damage is one of the targets for the antiox-
idant-based composite therapy, because lipid peroxidation is
implicated as a major step involved in the progression of
brain damage after ischemia/reperfusion.9—11) Several trials
thus have been carried out using a small molecular antioxi-
dant such as lipoic acid12) for the intervention of damage pro-
gression in the brain. However, the results were limited, prob-
ably because several steps occur in pathogenesis in the brain
after ischemia/reperfusion besides lipid peroxidation, such as
the release of excitable amine,13) Ca21,14) cytokines15) and
NO.16)
In the present studies, we focused our attention on one for-
mula of Chinese traditional medicine, Qizhu Tang (QZT).
QZT consists of four herbal components; Rhizoma atracy-
lodis macrocephalae, Poria, Radix notoginseng and Radix
astragali seu hedysari. It has been used to treat syndromes
related to Qi depression in the spleen and stomach such as
dyspepsia, water stool, lassitude of limbs, slow and weak
∗ To whom correspondence should be addressed. e-mail: konishi@niigata-pharm.ac.jp
pulse and pale complexion. It is also used to promote blood
circulation to remove blood stasis and has especially been
prescribed for enhancing the immune function of the body.17)
QZT can enhance celiac macrophage phagocytic function in
mice and enhances serum IgG in humans.18) It also facilitates
the restoration of thymus atrophy and hypofunction, which
are induced by malnutrition.19)
We previously observed the marked protective and repara-
tion effects of another Qi and immune stimulating Chinese
traditional medicine, Shengmai san (SMS), on cerebral is-
chemia/reperfusion damage in rats.20) Although the compo-
nent herbs are completely different from each other, the wide
spectrum of QZT effects, including its Qi and immune mod-
ulating activity, allow us to evaluate the protective effect of
QZT on brain oxidative injury after forebrain ischemia/reper-
fusion in rats as another example of antioxidant-based com-
posite formula.
MATERIALS AND METHODS
Chemicals and Herbal Materials 2,29-Azobis (2-
amidino-propane) dihydrochloride (AAPH), glutathione re-
duced form (GSH), 2-thiobarbituric acid (TBA) and 1,1-
diphenyl-2-picrylhydrazyl (DPPH) were purchased from
Wako Pure Chemical Ind. Disodium EDTA was obtained from
Kanto Chemical Co., Ltd., Sodium dodecyl sulfate (SDS)
from nacalai tesque Co., Ltd. b -NADPH and GSH reductase
from Sigma Co., Ltd., U.S.A. 5,5-Dimethyl-1-pyrroline-N-
oxide (DMPO) was purchased from LABOTEC Co., Ltd.,
Tokyo. All other chemicals were of analytical grade.
Dried herbal materials, including Rhizoma atractylodis
macrocephalae (B), Poria (F), Radix notoginseng (S), and
Radix astragali seu hedysari (H) were obtained from Magiya
Pharmacy Co., Ltd., Niigata, Japan. B, S and H are products
of Hei Long Jiang Sheng, Yun Nan Sheng and Si Chuan
Sheng in China, respectively. F is a product of Korea.
QZT and Their Related Preparations QZT was pre-
© 2001 Pharmaceutical Society of Japan
May 2001
pared by gently boiling a mixture of B (24 g), F (18 g), S
(36 g) and H (36 g) in 250 ml of distilled water for 30 min to
reduce the volume to 100 ml. The herb mixture was soaked
for 1 h at room temperature before boiling. The decoction
was then filtered through delipidated gauze and stored in a
refrigerator until use. The concentration of the decoction was
1.14 g dried herb mixture/ml. Similarly, the preparations con-
taining each QZT component alone or the combinations
lacking one or two components from the complete QZT for-
mula were decocted in the same way as above, using the
same amount of component herb(s) as in the QZT formula.
Hydroxyl Radical Scavenging Assay by the ESR Spin
Trapping Method Hydroxyl radical (· OH) scavenging ac-
tivity of QZT was determined by a spin trapping ESR using
DMPO as a spin trap reagent according to the method de-
scribed previously.20) Both the Fenton reaction and H2O2/UV
systems were used as the · OH source.
Comparative study on the · OH scavenging activity of QZT
and its related decoctions was carried out in the presence of
liver homogenate using the Fenton system as · OH generator.
Briefly, rat liver homogenate (0.923 mg protein) was previ-
ously incubated with an aliquot of QZT or its related decoc-
tions at 37 °C for 30 min. The homogenate (100 m l) was
mixed with 160 m l of distilled water, 30 m l of 100 mM H2O2
and 30 m l of 200 mM DMPO. Then, 30 m l of 2 mM FeSO4 was
finally added to the mixture to initiate the Fenton reaction.
The DMPO–OH adduct formation was measured in a silica
flat cell 2 min after the addition of FeSO4 using a JEOL JES-
TE 200 electron spin resonance (ESR) spectrometer (X-Band
Microwave Unit). The spectrometer settings were as follows:
microwave power, 8 mW; microwave frequency, 9.20 GHz;
modulation amplitude, 0.1 mT; time constant, 0.03 sec;
sweep time, 30 sec; center fields, 332.6/322.6 mT.
In Vitro Radical Scavenging Activity Determined by
DPPH Quenching The DPPH radical scavenging activity
of QZT and its related decoctions was determined according
to the method by Yoshida et al.21) QZT sample (0.1 ml) was
incubated with 0.2 ml of rat liver homogenate (0.923 mg as
protein) prepared in 1.15% (w/v) KCl for 30 min at room
temperature. Then, 0.1 ml of aqueous DPPH (1.531024 M)
was added, shaken vigorously, and allowed to stand for
30 min at room temperature. Unreacted DPPH was deter-
mined by the absorbance at 520 nm. For the control, physio-
logical saline solution was added instead of QZT sample.
In Vitro Lipid Peroxidation Assay by AAPH-Induced
TBARS Formation in Rat Liver Homogenate AAPH-ini-
tiated lipid peroxidation22) was determined by TBA reactive
substances (TBARS) formation in normal rat liver ho-
mogenate according to the method by Ohkawa et al.23)
Briefly, the reaction mixture containing 0.1 ml liver ho-
mogenate (0.923 mg as protein), 0.1 ml AAPH (1 mM aque-
ous solution), and 0.1 ml QZT and its related decoctions was
incubated aerobically at 37 °C for 30 min. Then, 0.2 ml of
8.1% SDS, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8%
aqueous TBA were added. The mixture was finally made up
to 4.0 ml with distilled water and heated at 95 °C for 60 min.
After adding 1.0 ml of distilled water, the solution was ex-
tracted with 5.0 ml of n-butanol and pyridine (15 : 1 v/v).
TBARS was determined by the absorbance at 532 nm in the
organic layer after centrifugation.
A Rat Model of Cerebral Ischemia-Reperfusion Male
559
Wistar rats (6 weeks old and 160—182 g body weight) pur-
chased from SLC Inc., Japan were conditioned for one day
by allowing free access to pelletted diet and water before the
experiment.
The forebrain ischemic treatment and other experimental
conditions were given previously.20) Briefly, a small incision
was made in the abdomen under anesthesia to expose the
duodenum and then QZT and its related decoctions were ad-
ministered directly into the lumen of the duodenum with a
syringe at a dose of 4 ml/rat 2 h before the cerebral ischemic
operation. Anesthesia was induced with diethyl ether and
maintained with pentobarbital. For saline control, rats were
administered physiological saline solution instead of a test
preparation. The normal control rats were not subjected to
any treatment.
For ischemic operation, a middle ventral incision was
made in the neck. Both right and left common carotid arter-
ies were exposed and occluded using nontraumatic aneurysm
clips. After 85 min of ischemia, the clips were removed to re-
store blood flow (reperfusion).
Brain Sample Collection and Biochemical Assay After
reperfusion for 45 min, the rats were decapitated under anes-
thesia. The whole brain was removed quickly, rinsed with
saline, and then frozen in a freezer (280 °C) until used. The
tissue was suspended in cold 0.05 M phosphate buffer con-
taining 1.15% (w/v) KCl (1 g wet tissue/9 ml), and homoge-
nized using a glass homogenizer at 0 °C.
TBARS was determined as described above but without
adding AAPH. GPX activity was determined according to
the method of Albrecht.24) Briefly, an aliquot of the brain ho-
mogenate (4 mg wet tissue) was mixed in a quartz cuvette
with 935 m l of the coupling solution containing 33.6 mg dis-
odium EDTA, 6.5 mg NaN3, 30.7 mg of GSH, 16.7 mg
NADPH and 100 units of GSH reductase in 100 ml of 50 mM
Tris–HCl (pH 7.6). Kinetic decay of NADPH fluorescence
(Ex. 355 nm/Em. 465 nm) was measured after the addition of
25 m l of 1 mM H2O2 as a substrate using a Hitachi model 650-
60 fluorescence spectrophotometer.
RESULTS
In Vitro Antioxidant Activities of QZT and Its Compo-
nents First, we examined · OH scavenging activity of QZT
by ESR using DMPO as a spin trap reagent. As shown in Fig.
1, the DMPO–OH signal produced by the Fenton reaction
was reduced by the addition of QZT in a concentration de-
pendent manner. The 50% inhibitory concentration (ID50) of
QZT for DMPO–OH formation was compared to that ob-
tained for Trolox as a reference radical scavenger. The QZT
decoction was found to have a strong · OH scavenging activ-
ity comparable to that by 0.03 mM Trolox. In order to know
whether the observed · OH scavenging activity of the QZT
was due to its direct scavenging of · OH or due to the inhibi-
tion of · OH generation in the Fenton reaction, the inhibition
of DMPO–OH formation by QZT was also examined using a
low concentration of DMPO as the spin trapping reagent
(2 mM; 1/10 of the above condition). The result was that the
dose-response curve shifted to the left without changing
shape significantly. The ID50 obtained was, however, approxi-
mately 1/6.4 of that observed with high DMPO concentration
(20 mM) (Fig. 2), indicating that QZT not only scavenges
560 Vol. 24, No. 5

Fig. 1. Effects of QZT on Fenton and H2O2/UV Mediated DMPO-OH Formation

QZT was prepared as described in the Methods section and the concentration was 1.14 g dry herb/ml decoction. (a) An aliquot of QZT in metal free distilled water was added
with an aliquot of 150 mM DMPO and 50 mM H2O2 to make the final volume of the reaction mixture to 300 ml, then the Fenton reaction was initiated by the addition of an aliquot of
FeSO4 stock solution to generate · OH. Final concentrations of DMPO, H2O2 and Fe21 were 20 mM, 10 mM and 0.1 mM, respectively. ESR spectra were determined 2 min after onset
of the · OH generation. (b) Hydroxyl radical was also generated by a UV/H2O2 system. The reaction mixture containing QZT, DMPO and H2O2 as above was illuminated with a
transilluminater (305 nm) for 1 min at room temperature, then the DMPO–OH signal was recorded 1 min after the reaction. Trolox concentration used as a reference was 0.05 mM.

Fig. 2. Effect of DMPO Concentration on QZT Dependent Inhibition of

DMPO–OH Formation
QZT preparation and spin trapping ESR conditions were the same as in Fig. 1. ID50
was determined using different DMPO concentrations in the Fenton reaction (d 20 mM
DMPO, s 2 mM DMPO). H2O2/UV system (n 20 mM DMPO).

· OH directly but also inhibits the · OH generation in the Fen-

ton reaction.25) This was further confirmed when the · OH
scavenging activity of QZT was determined in the H2O2/UV
system as · OH source. In this system, the Trolox equivalence
of the QZT decoction was calculated as approx. 0.1 mM.
Effect of QZT on Cerebral Oxidative Damage Induced
by Forebrain Ischemia/Reperfusion in Rats The experi-
mental conditions described in the Methods section were es-
sentially the same as our previous study on SMS which
showed marked prevention and repair of cerebral oxidative
damage after ischemia/reperfusion treatment.20) The whole
brain homogenate prepared from the brain removed after
45 min reperfusion was used for determining both TBARS
formation as an index of lipid peroxidation and glutathione
peroxidase (GPX) activity as an index of scavenging poten-
Fig. 3. Protective Effect of QZT on Cerebral Ischemia/Reperfusion Injury
in Rats
QZT decoction (4 ml) was administered into the rat duodenum 2 h before cerebral is-
chemia treatment. At 45 min reperfusion after 85 min ischemia, TBARS (a) and GPX
activity (b) were determined for the whole brain homogenate. Triplicate determinations
were done for one homogenate. Values are the mean of 3 rats with S.D. * p,0.05.
tial toward lipid peroxides to evaluate the oxidative damage
to the brain. As is shown in Fig. 3a, TBARS formation was
markedly increased in the saline control after the ischemia/
reperfusion (approx. 210% of normal control). However, in
the QZT treated rats, the TBARS formation in the damaged
brain was significantly inhibited. The TBARS level in the
damaged brain was increased only to 16% of the normal con-
trol. QZT thus inhibited approximately 86% of the ischemia/
May 2001
Fig. 4. Hydroxyl Radical Scavenging Activities of QZT Component and
Related Decoctions
DMPO–OH was determined by ESR as described in Fig. 1. QZT component and the
related decoctions were incubated with liver homogenate for 30 min at 37 °C before
ESR measurement. Data are give as an average of triplicate experiments with S.D.
reperfusion induced damage in the rat’s brain (n53, p,0.05).
At the same time, GPX activity was measured to evaluate
the defense potential of the cell against the oxidative stress.
A considerable loss of GPX activity was observed in the
saline control brain after ischemia/reperfusion (approx. 40%
of normal control was retained), but the loss of GPX activity
was markedly prevented by the QZT preadministration (Fig.
3b). The GPX activity was maintained at 80% of the normal
level in the QZT pretreated rats which corresponds to ap-
proximately 40% protection of the damage induced.
In Vitro Antioxidant Activities of QZT Component and
Their Combinations The · OH scavenging activities of
QZT components and their combinations in vitro were deter-
mined through the ESR spin trapping method in the presence
of rat liver homogenate with which the decoctions were
preincubated for 30 min at 37 °C (Fig. 4). The scavenging ac-
tivity of the HBS combination was the strongest among the
preparations tested and was comparable to that of the com-
plete QZT decoction. The HBF, HS and HB combinations
followed. Relatively weak but significant inhibition was also
found in H, B alone and in HFS, BS and HF combinations.
However, F did not show any scavenging activity and even
behaved as a prooxidant under the experimental conditions.
Likewise, the BF combination showed weak prooxidant ac-
tivity. The same trends were obtained when the scavenging
activities were determined in distilled water without liver ho-
mogenate (data not shown).
DPPH radical quenching activities were also examined in
vitro. Only complete QZT formula and HBS combination
showed marked quenching activity for DPPH radical (100
and 100.5%, respectively). No other combinations nor the
component alone showed significant quenching except HS
and S which showed moderate quenching activities (approxi-
mately 25 and 22%, respectively) (data not shown).
The inhibitory effects of QZT components and their com-
binations on AAPH-induced TBARS formation were further
evaluated in the rat liver homogenate. Complete QZT for-
mula showed the highest activity (100%), followed by the
HBS combination (approx. 79%). Considerable inhibition
was also achieved with B, S and HB (58, 58 and 49%, re-
spectively). Other decoctions showed only limited inhibition,
561
a)
b)
Fig. 5. In Vivo Protection of Ischemia/Reperfusion Induced Damage in
Rats by Decoctions Having High · OH Scavenging Potency
Experimental conditions are the same as in Fig. 3. Values are mean6S.D. (n56).
* and # are p,0.005 and p,0.05, respectively, compared to saline control.
and both the HBF and HF combinations even behaved as
prooxidant (data not shown).
In Vivo Antioxidant Activities of QZT Components and
Their Combinations To understand the rationale of the
composite formula in preventing the cerebral oxidative dam-
age, the effect on the ischemia/reperfusion damage in a rat
brain was examined for QZT and some of the QZT-related
decoctions which have shown as strong · OH scavenging ac-
tivity as QZT in vitro (Fig. 5). F was also examined as a ref-
erence because it did not show any · OH scavenging activity,
even enhanced DMPO–OH signal. The complete QZT for-
mula showed the most effective prevention of TBARS forma-
tion in the damaged brain followed by the HBS combination
(Fig. 5a). Although the HBS combination showed rather
stronger in vitro DPPH and · OH quenching activities than
QZT (approx. 101 and 109% of complete QZT, respectively),
the in vivo activity was only 46% of QZT. Likewise, the HBF
and HS combinations which also showed comparable · OH
scavenging activity to QZT did not significantly inhibit
TBARS formation in vivo. F was not effective at all. The
same trend was observed in their action on GPX activity in
the damaged brain. The most effective protection was at-
tained by QZT but not by other decoctions except HBS,
which showed a moderate protective effect (Fig. 5b).
DISCUSSION
Traditional Chinese herbal medicines have recently at-
562
Fig. 6. Relationship between · OH Scavenging and TBARS Inhibition Ac-
tivities among the QZT Related Decoctions in Vitro
The data showing prooxidant activity in Fig. 4 were removed. s, all preparations ex-
amined; j, preparations containing Poria; n, prepartions containing Radix Astragali.
tracted much attention as alternative medicines useful for
treating or preventing so-called lifestyle-related disorders.7)
Since the oxidative stress is implicated in the initiation
and/or propagation of these pathophysiologies,2) the antioxi-
dant potential is considered to be a reasonable index for eval-
uating the therapeutic effectiveness of Chinese herbal medi-
cines.
In the present experiments, we studied first the · OH scav-
enging activity of QZT because · OH is highly reactive and
considered to be a major causative factor of cellular oxidative
injuries.25) Indeed, the QZT decoction was found to have a
high potential of · OH scavenger comparable to 0.1 mM
Trolox when the scavenging activity was determined by
H2O2/UV as the · OH source. It was also determined that the
observed inhibitory action of QZT on DMPO–OH formation
in the Fenton system resulted from both the direct scavenging
of · OH and the inhibition of its generation (Fig. 2), probably
due to the masking effect on ferrous ion by QZT, as shown
elsewhere.26) Indeed, a much lower value was determined for
the Trolox equivalent activity of QZT (approx. 0.03 mM)
when determined through the use of the Fenton reaction, as
the · OH source indicating QZT was consumed not only for
scavenging · OH but also for reacting with Fe21.
It was further found that the QZT decoction administered
before ischemia/reperfusion effectively protects the oxidative
damages in the brain (Fig. 3), as was shown by another Chi-
nese herbal medicine, SMS.20) SMS is also a strong antioxi-
dant and has similar immune and Qi modulating functions as
QZT.
Chinese traditional medicines are usually formulated with
Vol. 24, No. 5
several herbal components having different physiological ac-
tivity. Therefore, it is known that the synergism among the
component herbs plays an important role in the physiological
action such that combination of two herbs enhances each
original function or decreases the toxic effect of the partner
herb.27) In the present study, we analyzed in vitro whether
such synergism also occurred among the component herbs of
QZT in terms of antioxidant activities.
When the · OH scavenging activity was determined for all
the QZT components and their combinations, H and B were
found considerably active followed by S. However, F en-
hanced DMPO–OH formation rather than decreased it (Fig.
4). This type of enhancement of DMPO–OH formation is re-
ported in several chemical systems involving ion chelate for-
mation as well as in our previous report on ferric iron/lactate
complex.28)
When each component herb was mixed with another, es-
sentially an additive effect of the component herb was ob-
served among the H, B, S in terms of · OH scavenging activ-
ity. However, F, which has a prooxidant nature itself, behaved
differently. For example, F stimulated the antioxidant activity
of HB but negatively affected on the HS and BS combina-
tions to reduce their activities. Therefore, HBS combination
showed the maximal · OH scavenging activity comparable to
complete QZT followed by the HBF and HS combinations.
When the · OH scavenging activity of each decoction was
correlated to other in vitro antioxidant parameters (TBARS
and DPPH quenching activities) in Fig. 6, the activity was
not correlated with either TBARS inhibitory activity or
DPPH quenching activity among the decoctions. In the fig-
ure, however, it was noticed that all the F containing decoc-
tions except complete QZT showed quite low TBARS in-
hibitory action regardless of their · OH scavenging potency.
Thus no correlation was established between TBARS and
· OH scavenging activities among the F containing decoc-
tions. In contrast, a rather good correlation was observed
among the decoctions containing H. These results suggest
that each component behaves differently in each antioxidant
assay system. Indeed, only the HBS combination showed the
strong antioxidant potential comparable to QZT in all three
antioxidant assays, but none of the other decoctions showed
this trend in their antioxidant potential in any of these three
in vitro assays (data not shown).
Therefore, we selected the decoctions that showed almost
the same · OH scavenging activity as QZT and examined
their protective effect on cerebral ischemia/reperfusion dam-
age in rats, together with F as a negative reference.
The most effective prevention of TBARS formation in vivo
was accomplished by complete QZT followed by the HBS
combination. However, the activity of the HBS combination
was only half that of complete QZT. Other combinations,
HBF and HS showed only limited activity. F did not show
any inhibitory action at all, as expected. GPX activity was
also preserved most effectively by complete QZT followed
by the HBS combination. However, the activity of HBS was
only 33% of QZT. All other preparations afforded significant
protective activity in vivo. This trend observed in in vivo ac-
tion was not consistent with any of the in vitro antioxidant in-
dications examined above. However, the present studies re-
vealed the propriety of the idea that Chinese traditional med-
icine is an interesting model for antioxidant-based combina-
May 2001
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26)
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