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Protective effects of Choto-san and hooks and stems of Uncaria sinensis against delayed neuronal death after transient forebrain ischemia in gerbil.

Abstract

Previously, we revealed that Choto-san (Diao-teng-san in Chinese), a Kampo formula, is effective on vascular dementia clinically, and the hooks and stems of Uncaria sinensis (OLIV.) HAVIL., a medicinal plant comprising Choto-san, has a neuroprotective effect in vitro. In the present study, for the purpose of clarifying their effects in vivo, we investigated whether the oral administration of Choto-san extract (CSE) or U. sinensis extract (USE) reduces delayed neuronal death following ischemia/reperfusion (i/rp) in gerbils. Transient forebrain ischemia was induced by bilateral carotid artery occlusion for 4 min, and two doses (1.0% and 3.0%) of CSE or USE were dissolved in drinking water and provided to the gerbils ad libitum from 7 days prior to i/rp until 7 days after i/rp. It was found that 1.0% and 3.0% CSE treatments significantly reduced pyramidal cell death in the hippocampal CA1 region at 7 days post i/rp. Three percent USE treatment also inhibited pyramidal cell death significantly at 7 days after i/rp. Superoxide anion and hydroxyl radical scavenging activities of the homogenized hippocampus at 7 days after i/rp in the 1.0% CSE- and 3.0% USE-treated groups were significantly enhanced compared to those of control. Further, lipid peroxide and N[O.sub.2.sup.-]/N[O.sub.3.sup.-] levels of the homogenized hippocampus at 48 h after i/rp in the 1.0% CSE- and 3.0% USE-treated groups were significantly lower than those of control. These results suggest that the oral administration of CSE or USE provides a protective effect against transient ischemia-induced delayed neuronal death by reducing oxidative damage to neurons.

[c] 2004 Elsevier GmbH. All rights reserved.

Keywords: Choto-san; Uncaria sinensis; Cerebral ischemia; Reperfusion; Hippocampus; Neuroprotection

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Introduction

The brain is particularly susceptible to an insufficiency of blood supply. Permanent or transient ischemia is a critical factor in the appearance of brain injury, and it is induced at least in part by the toxicity of reactive oxygen species (White et al., 2000; Bolanos and Almeida, 1999).

During brain ischemia, excessive amounts of glutamate are released from vesicles in pre-synaptic neurons, overstimulating glutamate receptors in post-synaptic neurons, especially N-methyl-D-aspartate receptors (White et al., 2000). This event leads to excessive [Ca.sup.2+] influx into neurons through voltage-dependent and more glutamate-regulated [Ca.sup.2+] channels (Lipton and Rosenberg, 1994), followed by the activation of the [Ca.sup.2+]-dependent neuronal form of nitric oxide (NO) synthase and the subsequent production of NO free radicals (NO*) (White et al., 2000). In the later phase of reperfusion, several stimuli involving activation of some kinds of cytokines strongly induce the [Ca.sup.2+]-independent inducible form of NO synthase (iNOS) expression in glial cells, allowing excessive and uncontrolled production of NO * (Bolanos and Almeida, 1999). In this process, large amounts of superoxide anions ([O.sub.2.sup.-]*) are generated as a result of mitochondrial dysfunction, accumulation of hypoxanthine (HPX), release of arachidonic acid, activation of leukocytes, and so forth (White et al., 2000). Increasing evidence from in vitro studies suggests that the endogenous formation of peroxinitrate anion (ONO[O.sup.-]), from the reaction of NO* with [O.sub.2], and further hydroxyl radical (HO*) generation, may be a possible mechanism by which neurotoxiciy is induced (Lafon-Cazal et al., 1993). Knowledge of these pathophysiological mechanisms has enabled investigators to develop successful therapeutic strategies against brain injury induced by ischemia/reperfusion (i/rp) in animal experiments, such as glutamate receptor antagonists (Ishimaru et al., 1997), NO synthase (NOS) inhibitors (Kohno et al., 1996), antioxidants/free radical scavengers (Bagenholm et al., 1996; O'Neill et al., 1997), and so on.

Choto-san (Diao-Teng-San in Chinese), a traditional Chinese/Japanese (Kampo) formula, has been administered to relatively aged patients with physical weakness and such subjective symptoms as headache, dizziness, vertigo, tinnitus, and so forth in Japan. Many of these symptoms are thought to originate from disorders in the cerebrovascular system. Recently, we demonstrated the effectiveness of Choto-san on patients with vascular dementia by well-controlled and double-blind studies (Shimada et al., 1994; Terasawa et al., 1997). Uncaria Uncus Cum Ramulus originating from the hooks and stems of Uncaria sinensis (OLIV.) HAVIL. is regarded as the main medicinal plant comprising Choto-san. We also recently revealed that the U. sinensis water extract and its phenolic and alkaloid compounds have protective effects against glutamate- and NO donor-induced neuronal death in cultured cerebellar granule cells (Shimada et al., 1998, 1999, 2001, 2002). However, the protective effect of the orally administered Choto-san or U. sinensis against brain injury induced by ischemia in vivo has not been sufficiently clarified.

It is well known that forebrain cerebral i/rp by transient occlusion of bilateral carotid arteries induces delayed neuronal death in the hippocampal CA1 region in the gerbil (Kirino, 1982). In the present study, using this animal model, we investigated the effects of orally administered water extracts of Choto-san and U. sinensis on delayed neuronal death in the hippocampal CA1 region, and also on [O.sub.2.sup.-]* and HO* scavenging activities and lipid peroxide (LPO) and nitrite (N[O.sub.2.sup.-])/nitrate (N[O.sub.3.sup.-]) production in the hippocampus.

Methods

Preparation of Choto-san and Uncaria sinensis extract

Choto-san was composed of 11 kinds of crude drugs mixed in the ratios shown in Table 1. All of these crude drugs were purchased from Tochimoto Pharmaceuticals (Osaka, Japan). This mixture (total 100 g) was extracted with boiling water (500 ml) for 50 min. The solution was centrifuged at 10,000g for 30 min, and the supernatant was then converted to freeze-dried powder as Choto-san extract (CSE). The water extracts of U. sinensis extract (USE) and other constituents of Choto-san were obtained from 100 g of each of the dried crude drugs by the above procedure. The yields of Choto-san and each of the constituent extracts are shown in Table 2.

Analysis of 3D-HPLC fingerprints of CSE and USE

CSE and USE (0.5 g) were extracted with methanol (20 ml) under ultrasonication for 30 min. The solution was filtrated and then submitted to HPLC analysis.

HPLC equipment was controlled with an HPLC pump (LC-10AD; Shimadzu, Kyoto, Japan) using a TSK-GEL, ODS-80TS column (4.6[phi] X 250 mm), and elution was done with solvents (A) 0.05 M AcON[H.sub.4] (pH 3.6) and (B) C[H.sub.3]CN. A linear gradient of 100% A and 0% B changing over 60 min to 0% A and 100% B was used. The flow rate was controlled with LC-10AD at 1.0 ml/min. The eluate from the column was monitored, and the three-dimensional data were processed with a diode array detector (SPD-M10A; Shimadzu, Kyoto, Japan). The three-dimensional HPLC charts of the methanol solutions of CSE and USE are shown in Figs. 1 and 2, respectively.

Animals

Adult male Mongolian gerbils (10 weeks old, 60-65 g) were purchased from Japan FUB Corporation (Hamamatsu, Japan). They were kept in an animal room at an ambient temperature of 23 [+ or -] 1[degrees]C under a 12-h dark-light cycle. They were allowed an adaptation period of at least 1 week. They were operated after 12 weeks (60-65 g). All animal use procedures were approved by the Committee on Animal Experimentation of Toyama Medical and Pharmaceutical University.

Grouping and treatment

Two doses (1.0% and 3.0%) of CSE were dissolved in drinking water and the animals were given access ad libitum. The animals were randomly divided into the following four groups: sham-operated group (sham), sham operation without CSE treatment; control group (control), i/rp without CSE treatment; 1.0% CSE group (1.0% CSE), i/rp with 1.0% CSE treatment; 3.0% CSE group (3.0% CSE), i/rp with 3.0% CSE treatment. CSE was administered to animals from 7 days prior to i/rp until 7 days after i/rp. The USE treatment protocol was essentially the same.

Surgery

Surgical procedures were performed according to the method of Kirino (1982) with slight modification. Gerbils were placed in an anesthetic container with 2.5% halothane and a mixture of nitrous oxide (50%) and oxygen (50%) for 5 min. During the surgical procedure, the anesthesic level was maintained with 1.0% halothane via a nose cone. The carotid arteries were exposed by a midline neck incision. After they were isolated, loosely looped around by silk threads, they were occluded for exactly 4 min with microaneurysm clips. Brain temperature was monitored by animal body temperature controller (ATB-1100, Nihon Kohden, Tokyo, Japan) with a temporalis muscle probe, and maintained at 37.0[degrees]C with a heating blanket and lamp. At the completion of the occlusion period, the clips were removed and reperfusion was visually confirmed before the neck was closed with silk threads. Sham surgeries were conducted in a similar manner, except that the carotid arteries were not occluded.

Histology

At 7 days after i/rp, the animals were deeply sedated by intraperitoneal injection of pentobarbital (0.1 ml, 65 mg/ml) and perfused with saline containing heparin (2 U/ml) followed by saline containing 10% paraformaldehyde. After perfusion, brains were removed, stored in paraformaldehyde solution for 7 days, and transferred to a solution containing 30% sucrose and 10% paraformaldehyde for 1 day. The brains were sliced into 20 [micro]m sections using a freezing microtome at -30[degrees]C, and the sections were mounted on slides and stained with cresyl violet. Stained slices at the level of the dorsal hippocampus 2.0 mm posterior to bregma were analyzed for damage to the CA1 region.

The numbers of viable cell bodies remaining in three portions of CA1 (medial, intermediate, and lateral) in both the left and right hemispheres of the brain were counted under a microscope (BH-2, Olympus, Tokyo, Japan) at X 400 magnification. Their total number was expressed as percentage of the average of sham-operated animals. The cell number was counted by an observer blinded to the various treatment groups.

[FIGURE 1 OMITTED]

Preparation of brain homogenate supernatant

Hippocampi were quickly separated from freshly removed brain samples and washed in ice-cold PBS (pH 7.4). They were then minced in ice-cold PBS and homogenized at a ratio of 1:10, w:v. After centrifugation of brain homogenates at 3000g for 10 min at 4[degrees]C, they were used for measurements of LPO, N[O.sub.2.sup.-]/N[O.sub.3.sup.-] and free radical scavenging activities against both [O.sub.2.sup.-] * and HO*.

Measurement of LPO

The LPO content in the homogenate supernatant of the hippocampus was measured by lipid peroxidation assay kit (Determina LPO; Kyowa Medex Co., Tokyo, Japan) according to the manufacturer's instruction.

Measurement of N[O.sub.2]/N[O.sub.3]

N[O.sub.2.sup.-]/N[O.sub.3.sup.-] content in the homogenate supernatant was measured by an automated system (ENO-10, EICOM CO., Kyoto, Japan), based on the Griess technique (Green et al., 1982).

Measurement of [O.sub.2.sup.-]* and HO* scavenging activities

Measurement of [O.sub.2.sup.-]* and HO* scavenging activities was performed as described previously (Ohsugi et al., 1999) with slight modification. After aliquots of the prepared hippocampal homogenate supernatant (50 [micro]l) were diluted to 10-fold with PBS (pH 7.4), [O.sub.2.sup.-]* and HO* scavenging activities of these samples were assessed by electron spin resonance (ESR) technique (Buettner, 1987).

In this experiment, [O.sub.2.sup.-]* was generated from a HPX-xanthine oxidase (XOD) reaction system in PBS (Mitsuta et al., 1990). Briefly, 15 [micro]l of 9.2 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (LABOTEC, Tokyo, Japan), 50 [micro]l of 2 mM HPX (Sigma, St. Louis, USA), 35 [micro]l of 5.5 mM diethylenetriamine-N,N,N',N'',N''-pentaacetic dianhydride (DETAPAC; Wako Pure Chemical Industries, Tokyo, Japan) and 50 [micro]l of prepared sample were put into a test tube. After adding 50 [micro]l of 0.4 U/ml XOD (Roche, Indianapolis, USA) and quick mixing, 200 [micro]l of the mixture was transferred to a flat quartz ESR cuvette, which was fixed to the cavity of an ESR spectrometer (JES-FR30, JOEL, Tokyo, Japan). Recordings of the spectra were made at 24[degrees]C and started at 1 min after the mixing of XOD; each scan took 1 min. Data were expressed as the ratio of the peak of the DMPO-OOH signal to the peak of the intrinsic standard, MnO (S/M). The scavenging activity (Scv) was calculated by the following equation:

[FIGURE 2 OMITTED]

Scv (%) = [[S/MBLANC - S/MSAMPLE]/[S/MBLANC]] X 100,

where S/MBLANC is the intensity of the ESR spectrum of DMPO-OOH spin adduct in PBS as a blanc, and S/MSAMPLE is the intensity of the ESR spectrum of DMPO-OOH spin adduct in the sample.

HO* was generated by the Fenton reaction (Kohno et al., 1991) consisting of 75 [micro]l of 0.1 mM [H.sub.2][O.sub.2], 50 [micro]l of each sample, 20 [micro]l of 92mM DMPO and 75 [micro]l of 0.1 mM FeS[O.sub.4]. The spectrum of DMPO-OH was measured at 1 min after the addition of [H.sub.2][O.sub.2]. Scavenging activity was calculated as described above.

In addition, the water extracts of Choto-san and its constituents were diluted to 0.2, 2.0, and 20.0 mg/ml with PBS. [O.sub.2.sup.-]* and HO* scavenging activities of each sample (50 [micro]l) were assessed by ESR technique, and then IC50 (inhibition concentration 50%) was calculated.

Statistical analysis

Values were expressed as mean [+ or -]S.E. The data were analyzed by one-way analysis of variance followed by Fisher's PLSD. A p-value <0.05 was considered statistically significant.

Results

In the present study, body weight did not differ among any of the groups at the time points of starting administration, surgery and sacrifice (data not shown). Water consumption also did not vary (data not shown), as all groups drank approximately 6 ml/animal/day (100 ml/kg/day) of water, allowing the calculation that the 1.0% and 3.0% CSE or USE groups ingested approximately 1.0 and 3.0 g/kg/day of CSE or USE, respectively.

[FIGURE 3 OMITTED]

Delayed neuronal death

In this animal model, neuronal damage occurred in the hippocampal CA1 region selectively, and no histological changes were observed in other areas of the brain.

Histological examination revealed that the number of viable pyramidal cells in the hippocampal CA1 region at 7 days after i/rp in the 1.0% and 3.0% CSE groups, administered CSE from 7 days prior to i/rp until 7 days after i/rp, were significantly greater than in control (Fig. 3). Similarly, those in this region at 7 days after i/rp in the 3.0% USE group, following the same administration schedule, were also significantly greater than in control (Fig. 4). Typical photographs of the hippocampi of these groups are shown in Fig. 5.

[O.sub.2.sup.-]* and HO* scavenging activities of homogenized hippocampus

We used the oral administration of 1.0% CSE and 3.0% USE in order to determine if they enhanced the [O.sub.2.sup.-]* and HO* scavenging activities in the hippocampus.

The [O.sub.2.sup.-]* and HO* scavenging activities of the homogenized hippocampus obtained from gerbils not undergoing i/rp after 7 days of continuous oral administrations of both of 1.0% CSE and 3.0% USE were significantly higher than those of non-treated control (Table 3).

The [O.sub.2.sup.-]* and HO* scavenging activities of the homogenized hippocampus at 7 days after i/rp in both 1.0% CSE and 3.0% USE groups, administered drugs from 7 days prior until 7 days after i/rp, were significantly higher than those of control (Table 4).

LPO and N[O.sub.2]/N[O.sub.3]

Both LPO and N[O.sub.2.sup.-]/N[O.sub.3.sup.-] levels in the homogenized hippocampus at 48 h after i/rp in both 1.0% CSE and 3.0% USE groups, treated from 7 days prior until 48 h after i/rp, were significantly lower than those of control (Fig. 6A and B).

[O.sub.2.sup.-]* and HO* scavenging activities (IC50) of extracts

Free radical scavenging activities (IC50) of CSE and each water extract of the crude drugs comprising Choto-san are shown in Table 2. CSE and USE have high scavenging activities against both [O.sub.2.sup.-]* and HO*. Other extracts, especially from Poria, Chrysanthemi flos and Zingiberis rhizoma, have relatively high scavenging activities against [O.sub.2.sup.-]* and HO*.

[FIGURE 4 OMITTED]

Discussion

In the present study, the oral administrations of CSE and USE to gerbils prevented delayed neuronal death of the hippocampal CA1 region induced by transient forebrain ischemia. This suggests that U. sinensis mainly contributes to the neuroprotective effect of Choto-san. Although we observed a tendency of higher-dose treatment to be more effective than lower-dose treatment, a distinctly dose-dependent effect was not evident. A ceiling effect might be present, although other concentrations need to be examined.

In general, it is well recognized that permanent or transient ischemia is a critical factor in the appearance of brain injury, and at least in part the toxicity of reactive oxygen species is responsible for the injury (White et al., 2000; Bolanos and Almeida, 1999). Consequently, antioxidants/free radical scavengers are considered to be effective therapeutic agents. Especially in natural products, it is known that phenolic compounds possess antioxidant and free radical scavenging properties (Plumb et al., 1998). An in vitro study showed that the phenolic compounds of red wine constituents can inhibit neuronal damage from the oxidative stress produced by NO generation (Bastianetto et al., 2000). In vivo studies have revealed that oral administrations of catechin (Inanami et al., 1998), green tea extract (containing catechin) (Hong et al., 2001) and Ginkgo biloba extract (containing ginkgo-flavonol glucosides, terpene lactones and procyanidines) (Calapai et al., 2000) provided protective effects against brain injury induced by transient forebrain ischemia in gerbils. U. sinensis also contains phenolic compounds, such as epicatechin, catechin, procyanidin B-1, procyanidin B-2, hyperin and caffeic acid (Shimada et al., 2001). Other investigators reported that Uncaria genus has antioxidant and free radical scavenging activities in vitro and in vivo. From in vitro experiments. Uncaria rhynchophylla (MIQ.) JACK., which contains similar components to U. sinensis, was reported to possess free radical scavenging activities when examined by ESR technique (Ohsugi et al., 1999; Liu and Mori, 1992). In vivo experiments using U. rhynchophylla showed increased antioxidant activity and inhibited lipid peroxidation in the brain of ferric chloride-induced epileptic rats (Liu and Mori, 1992), and also decreased the lipid peroxide level in the brain of kainic acid-induced epileptic rats (Hsieh et al., 1999).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

In the present study, it was revealed that [O.sub.2.sup.-]* and HO* scavenging activities of the hippocampal homogenate obtained from gerbils not only without i/rp procedure but also 7 days after i/rp were enhanced by oral administrations of 1.0% CSE and 3.0% USE. The data obtained from the cortex showed similar results (data not shown). In addition, the LPO levels of homogenized hippocampus in 1.0% CSE- and 3.0% USE-treated groups at 48 h post i/rp were lower than those of control. One of the mechanisms for these results being obtained may be free radical scavenging activities of CSE and USE themselves. Another may be the induction of endogenous antioxidants such as superoxide dismutase and glutathione peroxidase. Moreover, there are some possibilities that inhibition of NOS, an antagonistic effect on the glutamate receptors of neurons, and so on, might be involved. Further studies focusing on the clarification of these possible involvements need to be performed.

U. sinensis also contains alkaloid compounds, such as oxyindole alkaloids, corynoxeine, rhynchophylline, isorhynchophylline and isocorynoxeine, and indole alkaloids, geissoschizine methyl ether, hirsuteine and hirsutine (Shimada et al., 1999; Sakakibara et al., 1998). Rhynchophylline, isorhynchorhylline, corynoxeine, isocorynoxeine and hirsutine exhibit vasodilative and [Ca.sup.2+] channel blocking activity in isolated rat thoracic aorta (Horie et al., 1992). Hirsuteine non-competitively antagonizes nicotine-evoked dopamine release by blocking ion permeation through nicotinic receptor channel complexes in rat pheochromocytoma PC12 cells (Watano et al., 1993). Geissoschizine methyl ether decreases specific [.sup.3]H] 5-hydroxytryptamine binding to membrane preparations from rat brain (Kanatani et al., 1985). Further, the oral administration of geissoschizine methyl ether or hirsuteine inhibits glutamate-induced convulsion in mice (Mimaki et al., 1997). It was also reported that Uncaria genus, such as U. sinensis and U. rhynchophylla, has protective effects against neurotoxicity induced by excitatory amino acids in vivo (Mimaki et al., 1997; Hsieh et al., 1999) and in vitro (Shimada et al., 1998, 1999). Moreover, it was recently reported that intraperitoneal injection of methanol extract of U. rhynchophylla protected hippocampal CA1 neurons against transient forebrain ischemia in a 4-vessel-occlusion rat model, by anti-inflammatory effect such as inhibition of COX-2 expression (Suk et al., 2002). These reports suggest that, besides the antioxidative effect, several other effects of U. sinensis, such as [Ca.sup.2+] channel and receptor blocking, together with anti-inflammatory effect, might contribute to its neuroprotective ability.

In our recent in vitro study, USE exerted a protective effect against NO donor-induced neuronal death in cultured cerebellar granule cells (Shimada et al., 2002), suggesting that USE works in a protective manner against NO-medicated neurotoxicity. In the present study, we observed a reduction in N[O.sub.2.sup.-]/N[O.sub.3.sup.-] levels in homogenized hippocampi from CSE- and USE-treated groups at 48 h post i/rp. From this, we can suppose that CSE and USE might exert an inhibitory effect on the induction of iNOS production or its activation, although it may also result from an anti-inflammatory effect by a reduction of the initial oxidative damage. Further studies will need to focus on the effect of USE and CSE on iNOS induction.

We also recently demonstrated that phenolic and alkaloid fractions/compounds of USE suppressed vasocontraction induced by oxidative stress (Goto et al., 2000), and we showed that the oral administration of USE had a protective effect on the endothelial function of spontaneously hypertensive rats (Goto et al., 1999). Taking these profiles of U. sinensis together, we are able to make a convincing argument about its beneficial effects on the pathophysiological mechanisms of brain ischemia.

In conclusion, the results of the present study seem to indicate that the oral administration of CSE or USE produces a neuroprotective effect against i/rp-induced brain injury possibly by the free radical scavenging effect. This and the previously revealed beneficial effects of Choto-san and U. sinensis on nervous and vascular systems strongly point to their potential use for the prevention of the development of ischemic cerebral diseases.
Table 1. The 11 crude drugs composing Choto-san and their weight ratios

 Weight (g)

Uncariae Uncis Cum Ramulus Hooks and stem of Uncaria
 sinensis Haviland 3.0
Aurantii Nobilis pericarpium Peel of Citrus unshu Markovich 3.0
Pinelliae tuber Tuber of Pinellia ternata
 Breitenbach 3.0
Ophiopogonis tuber Root of Ophiopogon japonicus
 Ker-Gawler 3.0
Poria Sclerotium of Poria cocos Wolf 3.0
Ginseng radix Root of Panax ginseng C.A.
 Meyer 3.0
Chrysanthemi flos Flower of Chrysanthemum
 morifolium Ramatulle 3.0
Saphoshnikoviae radix Root and rhizome of
 Saposhnikovia divaricata
 Schischkin 3.0
Glycyrrhizae radix Root of Glycyrrhiza uralensis
 Fisher 1.0
Zingiberis rhizoma Rhizome of Zingiber officinale
 Roscoe 1.0
Gypsum Fibrosum CaS[o.sub.4] * 2[H.sub.2]O 5.0

Table 2. Yields and [O.sub.2.sup.-]* and HO* scavenging activities of
extracts of Choto-san and the comprising crude drug

 Yield of IC50 (mg/ml)
 extract (%) [O.sub.2.sup.-]* HO*

Choto-san 10.6 0.84 4.68
Uncariae Uncis Cum 7.0 0.02 2.77
Ramulus
Aurantii Nobilis 8.8 1.72 3.83
pericarpium
Pinelliae tuber 1.6 40.78 4.22
Ophiopogonis tuber 38.9 137.28 64.06
Poria 0.5 4.37 0.77
Ginseng radix 10.1 302.41 2.17
Chrysanthemi flos 6.2 0.55 4.12
Saphoshnikoviae radix 7.6 3.99 3.54
Glycyrrhizae radix 16.0 3.40 4.43
Zingiberis rhizoma 3.3 1.33 0.97
Gypsum Fibrosum 0.9 -- 2.00

Table 3. [O.sub.2.sup.-]* and HO* scavenging activities of homogenized
hippocampus obtained from gerbils without ischemia/reperfusion after 7
days of Choto-san extract (1.0% CSE) and 3.0% Uncaria sinensis extract
(3.0% USE)

 Control 1.0% CSE

[O.sub.2.sup.-]* 27.1 [+ or -] 1.0 42.6 [+ or -] 1.1**
scavenging
activity (%)
HO* 10.8 [+ or -] 3.2 24.4 [+ or -] 1.4**
scavenging
activity (%)

 3.0% USE

[O.sub.2.sup.-]* 34.6 [+ or -] 2.7**
scavenging
activity (%)
HO* 22.3 [+ or -] 2.7**
scavenging
activity (%)

Mean [+ or -] S.E., n = 8, ** p<0.01 compared with control.

Table 4. [O.sub.2.sup.-]* and HO* scavenging activities of homogenized
hippocampus obtained from gerbils 7 days after ischemia reperfusion of
Choto-san extract (1.0% CSE) and 3.0% Uncaria sinensis extract-treatment
groups (3.0% USE)

 Control 1.0% CSE

[O.sub.2.sup.-]* 32.6 [+ or -] 2.1 44.1 [+ or -] 1.4**
scavenging
activity (%)
HO* 13.8 [+ or -] 1.5 20.4 [+ or -] 1.0**
scavenging
activity (%)

 3.0% USE

[O.sub.2.sup.-]* 41.1 [+ or -] 1.3**
scavenging
activity (%)
HO* 23.0 [+ or -] 1.9**
scavenging
activity (%)

Mean [+ or -] S.E., n = 8, ** p<0.01 compared with control.


Acknowledgements

This work was supported by a Health Sciences Research Grant for Comprehensive Research on Aging and Health from the Japanese Ministry of Health, Labour and Welfare.

Received 22 November 2002; accepted 15 April 2003

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Koichi Yokoyama (a,*), Yutaka Shimada (a), Etsuro Hori (b), Nobuyasu Sekiya (a), Hirozo Goto (c), Iwao Sakakibara (d), Hisao Nishijo (b), Katsutoshi Terasawa (a)

(a) Department of Japanese Oriental Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan

(b) Department of Physiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan

(c) Department of Kampo Diagnostics, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan

(d) Pharmacognosy and Medical Resources Laboratory, Tsumura and Co., Amimachi, Ibaraki, Japan

*Corresponding author. Tel.: +81-76-434-7393; fax: +81-76-434-0366.

E-mail address: yokomac@ms.toyama-mpu.ac.jp (K. Yokoyama).
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Author:Yokoyama, Koichi; Shimada, Yutaka; Hori, Etsuro; Sekiya, Nobuyasu; Goto, Hirozo; Sakakibara, Iwao; N
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Sep 1, 2004
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