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Antagonistic effects of two herbs in Zuojin Wan, a traditional Chinese medicine formula, on catecholamine secretion in bovine adrenal medullary cells.

ABSTRACT

In order to research the target of superior efficacy and lesser side effects, combination of herbal materials has been applied to phytotherapy for thousands of years in China and some other countries. Zuojin Wan (ZJW), a famous traditional Chinese medicine formula, is used in treating gastric diseases in China. It is composed of two herbs, Rhizoma Coptidis (RC) and Fructus Evodiae (FE) in the ratio of 6: l(w/w). In the present study, we examined the effects of ZJW, RC, FE and active components isolated from these herbs on catecholamine (CA) secretion and intracellular calcium ([[[Ca.sup.2+]].sub.i]) in cultured bovine adrenal medullary cells. Extracts of ZJW and RC and berberine, palmatine and jatrorrhizine, components of RC, all inhibited CA secretion and rise in [[[Ca.sup.2+]].sub.i] induced by acetylcholine (ACh), veratridine (Ver) and/or 56 mM [K.sup.+]. On the other hand, extract of FE, evodiamine and rutaecarpine, components of FE, stimulated CA secretion and rise in [[[Ca.sup.2+]].sub.i] induced by ACh. Furthermore, different proportions of RC and FE caused different responses in CA secretion. The present findings suggest that two herbs in ZJW have opposite effects, i.e., inhibitory effect of RC and stimulatory effect of FE, on CA secretion induced by acetylcholine in cultured bovine adrenal medullary cells.

Keywords: Catecholamine Fructus Evodiae Rhizoma Coptidis Berberine Evodiamine Bovine adreanl medullary cells

Introduction

For a long period of time, the pharmaceutical industry has been searching for a single drug that specifically targets a single disease-causing molecule. While the pathogenesis of disease is so complex, the therapeutic effect of single drug may be modest and hampered by various side effects or resistances of drugs in clinic. On the contrast, there are numerous herbal medicines that are considered to be potential agents for their satisfactory curative efficacies with few side effects in traditional Chinese medicine (TCM). It is believed that combination of multiple herbs under the guidance of theories of TCM, called formulae, could hit multiple targets and reduce the adverse effects. For instance, it is reported that the combination of Realgar (tetraarsenic tetrasulfide), Indigo Naturalis, Radix Salviae Miltiorrhizae and Radix Pseudostellariae had exerted a synergic effect in treating human acute promyelocyte leukemia (APL) (Wang et al. 2008). The concept of herbal combination in phytotherapy has been paid more and more attentions (Wagner 2006; Li and Zhang 2008; Ma et al. 2009).

Zuojin Wan (ZJW) is a famous formula of TCM used for treatment of gastric diseases. It is composed of two herbs, Rhizoma Coptidis (RC) and Fructus Evodiae (FE) in the ratio of 6: l (w/w). It was first recorded in Danxi Xinfa, a famous ancient medicine treatise written hundreds of years ago. Chemical investigations have shown that the active compounds are berberine, palmatine, jatrorrhizine in RC (Sheng et al. 2006). Evodiamine and rutaecarpine were active compounds in FE (Yang et al. 2009) (Fig. 1). ZJW was applied to treat gastrointestinal diseases for its properties of stopping vomiting, decreasing gastric acid and relieving pain. Recent studies revealed that ZJW has various beneficial effects, such as anti-inflammation, anti-ulcer and anti-acid activities and inhibitory effect on the growth of Heliobacter pylori (Chen et al. 2003).

[FIGURE 1 OMITTED]

Stress is believed to be an important cause of hyperacidity and ulceration. Previous studies have shown that long-term stress induces an excessive CA secretion which may be an important etiological factor of a variety of changes occurring in the pathogenesis of gastrointestinal diseases (Artz and Fitts 1996). A case report showed that there is an unequivocal relationship between stressful events with increased CA release and subsequent severe symptomatic gastrointestinal ischemia which play a pivotal role in the pathogenesis of gastric ulcer (Veenstra et al. 2007). It has also been elucidated that adrenergic system is involved in gastric secretion and pathogenesis of stress-induced ulcer (Sutoo et al. 1998). Accumulating evidence have suggested that decreasing of mucosal blood flow level results in ischemia and hypoxia which is one of causes for the formation of stress gastric ulcer (Guth 1979; Mitchell 2004 ).

Chromaffin cells of the adrenal medulla are derived from multipotent neural crest cells in the developing embryo. They share a common sympathoadrenal progenitor with sympathetic neurons. Thus, adrenal chromaffin cells are considered to be the sympathetic post-ganglionic neurons and have become specialized in the release of CA to the circulating blood flow. Bovine adrenal chromaffin cells have been widely used as a model system for studying the effects of drugs on CA secretion (Mao et al. 2008; Park et al. 2003). In these cells, our previous studies have shown that both ACh-induced [Na.sup.+] influx through nicotinic ACh receptor-ion channels and Ver-induced [Na.sup.+] influx via voltage-dependent [Na.sup.+] channels increase [Ca.sup.2+] influx via activation of voltage-dependent [Ca.sup.2+] channels, a prerequisite for secretion and synthesis of CA. In contrast, high [K.sup.+] directly gates voltage-dependent [Ca.sup.2+] channels to increase [Ca.sup.2+] influx without increasing [Na.sup.+] influx (Wada et al. 1985). Adrenal medullary cells have been a good model for detailed study of the actions of different drugs on CA secretion (Shinohara et al. 2007).

A previous study reported that berberine and palmatine, components of RC, inhibited catecholamine biosynthesis by modulation of tyrosine hydroxylase activity in PC12 cells (Lee and Kim 1996; Lee et al. 1996; Shin et al. 2000). Yoshizumi et al. (1997) also showed that evodiamine, an active compound extracted from FE, stimulated CA secretion from perfused bovine adrenal medulla, although they did not show any mechanism for it. The potential roles of RC and combination of RC and FE in catecholamine secretion have not been studied. In the present study, we examined the effects of ZJW, RC, FE and their components on CA secretion and rise in [[[Ca.sup.2+]].sub.i] in cultured bovine adrenal medullary cells. We found that ZJW, RC and their components inhibited CA secretion and rise in [[[Ca.sup.2+]].sub.i] induced by ACh, whereas FE and its components stimulated these responses. It seems that the combination ratio of RC versus FE plays an important role in the regulation of catecholamine secretion in the cells.

Materials and methods

Reagents and materials

Oxygenated Krebs-Ringer phosphate (KRP) buffer was used throughout. Its composition is as follows (in mM): 154 NaCl, 5.6 KCl, 1.1 [MgSO.sub.4],. 2.2 [CaCl.sub.2], 0.85 [NaH.sub.2][PO.sub.4], 2.15 [Na.sub.2][HPO.sub.4] and 10 glucose, adjusted pH to 7.4. Reagents were obtained from the following sources: Eagle's minimum essential medium (MEM) and new born calf serum from Gibco (USA); collagenase from Nitta Zerachin (Osaka, Japan); acetylcholine and veratridine from Sigma (St. Louis, MO, USA); and other chemicals used were of reagent grade obtained from Tianjin (Tianjin, China).

Preparation of the extracts for Chinese herbs

Chemical standards for berberine, palmatine, jatrorrhizine, evodiamine and rutaecarpine were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purities of the standards were all above 98%. 350 g Fructus Evodiae was decocted three times with 5600 ml water and for 0.5 h each time. The extracts of three times were combined, concentrated, and then dried in vacuum. 400 g Rhizoma Coptidis were decocted three times with 4800 ml water and for 1.5 h each time. The extracts were combined, concentrated, and then dried in vacuum.

Quantitative analysis of various active compounds in the extracts of RC and FE

For quantitative results of alkaloids in Rhizoma Coptidis, the HPLC analyses were performed on an Agilent 1100 series HPLC instrument (Agilent, Waldbronn, Germany) composed of a vacuum degasser, a quaternary pump, an autosampler, a column compartment, and a diode array detector (DAD). The chromatographic separation was carried out on a Waters Symmetry C18 column (5 [micro]m, [empty set] 3.9 mm x 100 mm) by setting the column temperature at 30[degrees]C. The mobile phase consisted of acetonitrile (A) and water containing 0.05% formic acid (B). A gradient program was used as follows: 12-16% A at 0-10 min, 16-16% A at 10-13 min, then 12% A hold for 7 min. The flow rate was kept at 0.8 ml/min. The detection wavelength was set to monitor at 345 nm. HPLC-UV chromatogram of standard substances and Rhizoma Coptidis sample are shown in Fig. 2A. Quantitative results of coptisine, jatrorrhizine, berberine and palmatine in Rhizoma Coptidis were 4.67%, 1.02%, 19.42% and 1.98%, respectively.

[FIGURE 2 OMITTED]

For quantitative measurement of alkaloids in Fructus Evodiae, reverse HPLC system with a Waters symmetryshield[TM] RP18 ([empty set] 3.9 x 150 mm, 5 [micro]m) column was used. The mobile phases were 50% acetonitrile:50% 5 mM phosphate solution at a flow rate of 1 ml/min according to published methods (Zhu 2007). Quantitative results of evodiamine and rutaecarpine in FE were 1.99% and 1.69%, HPLC-UV chromatogram of standard substances and sample are showed in Fig. 2B. In present study, ZJW consists of 77.2 g [ml.sup.-1] RC extracts and 12.8 g [ml.sup.-1] FE extracts. All the indicated materials were dissolved in water prior to use.

Isolation of bovine adrenal medullary cells and primary culture

Bovine adrenal glands were obtained from the city slaughterhouse. Bovine chromaffin cells were isolated following published methods (Mao et al. 2009). To examine the effects of ZJW and the active compounds from RC and FE on CA secretion, the cells were cultured at a density of 5 x [10.sup.5] cells/well in modified Eagle's MEM medium supplemented with 10% new born calf serum, 50 U/ml penicillin, and 50 [micro]g/ml streptomycin under a humidified atmosphere of 5% [CO.sub.2] and 95% air at 37[degrees]C in 24-well plates. The cells used for experiments were between 2 and 7 days of culture.

CA secretion from cultured bovine adrenal medullary cells

Chromaffin cells were washed three times before experiments. Then cells were incubated in the presence or absence of various secretagogues at 37[degrees]C for 10 min. The incubation medium was transferred immediately to a test tube containing perchloric acid (PCA) (final concentration, 0.4 M). CA (norepinephrine and epinephrine) secreted into the medium were adsorbed onto aluminum hydroxide and were directly injected into a HPLC system coupled with an electrochemical detector (HPLC-ECD).

Chromatographic conditions of HPLC-ECD method to detect the CA secretion from cultured bovine adrenal medullary cells

The chromatographic system consisted of an Agilent 1100 system with an electrochemical detector. A RP18 analytical column (250 x 4.6, [empty set] = 5 [micro]m) was used and separation was made by using an isocratic solvent system consisting of 89% phosphate buffer (containing 13.6 g [KH.sub.2][PO.sub.4], 2.5 g sodium-octanesulfoacid and 0.036 g EDTA, adjust pH to 3.3 with [H.sub.3][PO.sub.4]), 10% methanol and 1% acetonitrile. A flow rate of 1 ml/min was maintained and separations were conducted at 35[degrees]C. The injection volume was 20 [micro]l. The detection was obtained using an electrochemical detector with a potential of +0.7 mV.

Measurements of intracellular concentration of [Ca.sup.2+]

[Ca.sup.2+] mobilization was studied using a Fluorometric Imaging Plate Reader, Flexstation III [Molecular Devices, USA]. Cells were cultured in black-walled clear-base 96-well plates (Greiner, USA) at a density of 50,000 cells per well in culture media and grew for approximately 24 to 36 h in a 37[degrees]C [CO.sub.2] incubator. In Flex experiments, the cells were incubated with the Calcium 4 reagent (Molecular Devices, Sunnyvale, CA) for 60 min in a 37[degrees]C [CO.sub.2] incubator. The Calcium 4 reagent-loaded cells were then placed into the Flexstation III to monitor cell fluorescence ([lambda]ex=485 nm, [lambda]em=525 nm). Extracts and active compounds of ZJW, RC and FE were dissolved in assay buffer (final DMSO concentration kept below 1%) on 96-well plates and added to the loaded cells by the automated pipettor in the Flexstation III. Intracellular [Ca.sup.2+] mobilization responses were measured as relative fluorescence units (RFU) and expressed as the percentage of RFU between each group and control.

Statistics

All experiments were performed in triplicate and each experiment was repeated at least three times. Data are presented as means [+ or -] SD. The significance of differences between means was analyzed using one way ANOVA. When a significant F value was found by ANOVA, Dunnett's tests for multiple comparisons were used to identify differences among the groups. Values were considered statistically significant when P were less than 0.05. Statistical analyses were performed using SPSS 11.5 software.

Results

Effects of ZJW and its active compounds on various secretagogue-induced CA secretion in cultured bovine adrenal medullary cells

We examined the effects of extracts of ZJW, RC and FE and their active components on CA secretion induced by ACh (300 [micro]M), Ver (100 [micro]M) or 56 mM [K.sup.+]. 90 [micro]g/ml ZJW treated the cells for 10 min significantly inhibited CA secretion induced by ACh to 79.5% compared with ACh alone, while it showed no effect on CA secretion induced by Ver and high [K.sup.+] (Fig. 3A). Interestingly, extract (78.2 [micro]g/ml) of RC. inhibited the secretion of CA induced by ACh to 67.9%. The inhibition of CA secretion by the extract of RC seems to be stronger than that caused by ZJW (Fig. 3B). We also observed that berberine, palmatine and jateorhizine, inhibited CA secretion induced by ACh, Ver and/or 56mM [K.sup.+] (Figs. 3D, E, F). On the other hand, extract of FE (12.8 [micro]g/ml) significantly enhanced CA secretion induced by ACh, but not by Ver and 56 mM [K.sup.+] (Fig. 3C). We next examined the effects of evodiamine and rutaecarpine, active components of FE, on CA secretion induced by various secretagogues (Figs. 3G, H). They significantly stimulated CA secretion induced only by ACh.

[FIGURE 3 OMITTED]

Effects of different proportions of RC and FE crude extracts on CA secretion and rise in [[[ca.sup.2+]].sub.i] induced by ACh

To study the effects of different proportions of RC versus FE on CA secretion, we designed the following different combination proportions of RC versus FE (in [micro]g/ml): 77.2:12.8, 45:45, 36:54, 30:60, 22.5:67.5, 12.8:77.2. The ratio of RC versus FE (6:1) significantly inhibited ACh-induced CA secretion. When combination proportion of RC versus FE was increased, any significant increase in CA secretion induced by ACh was not observed. To the contrast, the ratio (l:6) of RC versus FE caused a significant increase in ACh-induced CA secretion, compared with ACh alone (Fig. 4A).
ACh            +   +    +   +   +      +         +

RC([micro] g/ml)  -  77.2  45  36  30  22.5  12.8
EF([micro] g/ml)  -  12.8  45  54  60  67.5  77.2

Fig.4. Effects of the different proportions RC and FE (A) and the raise
of [[[Ca.sup.2+]].sub.i] (B) induced by 300 [micro]M ACh. Data are means
+SD from three to four separate experiments carried out in triplicate
** P < 0.01 and * P < 0.05, compared with ACh alone.

Note: Table made from bar graph.


It's known that the raise of intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) is a prerequisite for CA secretion. Hence, we examined the effects of RC (78.2 [mu]g/ml) and FE (12.8 [mu]g/ml) and their combination on rise in [[[Ca.sup.2+]].sub.i] induced by ACh (300 [mu]M). As shown in Fig. 4B, RC (78.2 [mu]g/ml) plus FE (12.8 [mu]g/ml), combination of ZJW. and RC (78.2 [mu]/ml) significantly inhibited the raise of [[[Ca.sup.2+]].sub.i] induced by ACh.

Effects of berberine, palmatine and jateorhizine on various secretagogues-induced CA secretion and rise in [[[Ca.sup.2+]].sub.i]

Treatment of cells with berberine, palmatine and jateorhizine, components of RC, at 1 or 10-100 [micro]M significantly reduced ACh-induced CA secretion ([IC.sub.50]=9.92, 55.5, and 114 [micro]M, respectively) (Figs. 5A, B, C) and rise in [[[Ca.sub.2+]].sub.i] (Figs. 5D, E, F). Berberine, palmatine and jateorhizine also inhibited CA secretion ([IC.sub.50]=86.8, 94.6 and 106.2 [micro]M) (Figs. 6A, B, C) and rise in [[[Ca.sup.2+]].sub.i] induced by Ver (Figs. 6D, E, F). Furthermore, berberine and palmatine inhibited CA secretion ([IC.sub.50]=106 and 106.3 [micro]M, respectively) (Figs. 7A, B) and rise in [[[Ca.sup.2+]].sub.i] induced by 56 mM [K.sup.+] (Figs. 7C, D) in a concentration-dependent manner similar to that of CA secretion induced by 56 mM [K.sup.+]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

Effect of evodiamine and rutecarpine on ACh-induced CA secretion and the raise of [[[Ca.sup.2+]].sub.i]

Treatment of cells with evodiamine and rutecarpine, components of FE, at 100 [micro]M for 10 min significantly increased ACh (300 [micro] [micro]M)-induced CA secretion (Figs. 8A, B). Evodiamine and rutecarpine also increased the rise in [[[Ca.sup.2+]].sub.i] induced by ACh (Figs. 8C. D).

[FIGURE 8 OMITTED]

Discussion

Dual effects of ZJW and its components on CA secretion induced by ACh

In the present study, we demonstrated that ZJW inhibit ACh-induced secretion of CAs in cultured bovine adrenal medullary cells (Fig. 3). ZJW, a formula of traditional Chinese herb medicine, consists of two herbs, RC and FE, in the ratio of 6:1 (w/w). RC also inhibited CA secretion induced by ACh, whereas FE enhanced it in the cells. The mixture of RC and FE in the ratio of 6:1 significantly inhibited CA secretion induced by ACh (Fig. 4A). When the ratio of RC was decreased, the inhibitory effect of the two herb mixture was diminished. The mixture of RC and FE in the ratio of 1:6 (w/w) contrarily enhanced CA secretion induced by ACh.

In the present study, the percentage of berberine, palmatine and jatrorrhizine in RC was 14.6%, 5.1 % and 1.9%, respectively, and that of evodiamine and rutaecarpine in FE was 2.0% and 1.7%, respectively. Berberine, palmatine and jatrorrhizine, the components of RC, all inhibited CA secretion induced by ACh, veratridine and/or 56 mM [K.sup.+] (Figs. 5, 6 and 7). On the other hand, evodiamine and rutaecarpine, the components of FE, both stimulated ACh-induced CA secretion (Fig. 8). From these results, it seems that ZJW has dual effects on ACb-induced CA secretion, i.e., inhibitory effect of RC and stimulatory effect of FE on CA secretion.

Mechanism of ZJW, RC, FE and their components in the modulation of CA secretion

Our previous study (Wada et al. 1985) reported that [Na.sup.+] influx via activation of nicotinic acetylcholine receptor-ion channels and voltage-dependent [Na.sup.+] channels stimulates [Ca.sup.2+] influx through voltage-dependent [Ca.sup.2+] channels and evokes the secretion of CA in adrenal medullary cells. On the other hand, 56 mM [K.sub.+], which activates voltage-dependent [Ca.sup.2+] channels by membrane depolarization, also stimulates CA secretion directly by stimulate [Ca.sup.2+] influx through voltage-dependent [Ca.sup.2+] channels. Some studies also demonstrated that the increase of [[[Ca.sup.2+]].sub.i] through voltage-dependent [Ca.sup.2+] channels is a prerequisite for secretion and synthesis of CA (Tsutsui et al. 1996).

Therefore, we further examined the effects of the main components in ZJW, RC and FE on the rise in [[[Ca.sup.2+]].sub.i] induced by various secretogogues in adrenal medullary cells. As shown in Fig. 4B, ZJW and RC inhibited ACh-induced rise in [[[Ca.sup.2+]].sub.i] in association with CA secretion. Berberine, palmatine and jatrorrhizine, components of RC, all inhibited ACh-induced increase of [[[Ca.sup.2+]].sub.i] in a concentration-dependent manner similar to that of CA secretion (Fig. 5). These findings are consistent with previous reports showing inhibitory effects of protoberberine and palmatine on catecholamine biosynthesis (Lee and Kim 1996) and tyrosine hydroxylase activity (Lee et al. 1996) in PC12 cells, respectively. As shown in Fig. 3. FE had little effect on CA secretion induced by the secretagogues except for ACh. Therefore, we focused on the effects of evodiamine and rutecarpine on ACh-induced CA secretion. We found that evodiamine and rutecarpine significantly stimulate CA secretion as well as the rise in [[[Ca.sup.2+]].sub.i] induced by ACh (Fig. 8). To the best of our knowledge, this is the first direct evidence that ZJW, RC, FE and their components exert dual actions on CA secretion probably through various ion channels in adrenal medullary cells.

Pharmacological significance of ZJW, RC and FE-induced modulation of CA secretion

It is believed that stressors including social, behavioral and psychological factors play important roles in the development of human gastric ulcer disease (Dipiro et al. 1997); its pathogenesis is mainly associated with atrophy or a decline in mucosal resistance, or both. Previous studies reported that the production of CAs is stimulated via activation of the sympathetic adrenal axis under various stressful situations such as emotional excitation (Westfall and Westfall 2005). Excessive CA may lead to the contraction of gastric mucosal blood vessels, and then result in inhibition of mucosal blood flow, which, in turn, induces ischemia and hypoxia and subsequently causes the formation of gastric ulcers (Maev et al. 2007; Laine et al. 2008). On the basis of this hypothesis, our results may partly explain the pharmacological mechanism by which RC have protective effects on gastric ulcers, i.e., RC, one herb component of ZJW, could reduce side effects induced by FE, at least, in the respect of CA secretion. This idea of the balanced actions by RC and FE, two herbs of ZJW, is well supported by other traditional Chinese herb medicine. For example, it has been well-known that Radix et Rhizoma Tripterygii is a traditional Chinese herb which is used in treatment of arthritis in China. Some active components (such as triptolide and celastrol) in this herb have side effects which hampered the clinical use of Radix et Rhizoma Tripterygii. Nevertheless, combination of Radix et Rhizoma Tripterygii and Radix Glycyrrhizae Uralensis reduced the side effects induced by Radix et Rhizoma Tripterygii [Zhang et al. 2007).

In the present study, we showed that ZJW, a mixture of RC and FE, causes a mild inhibition of CA secretion induced by ACh. Because excessive CAs are harmful for the gastrointestinal system and RC has been used in clinic for thousands of years in China, one may question why ancient Chinese people used FE in addition to RC for the treatment of ulcers. Pharmacological studies reported that RC shows vasorelaxing and antiproliferative effects; it inhibits the growth of Heiiobacter pylori (HP) and the intestinal parasite Blostocystis hominis in vitro (Yang et al. 1996; Zhang et al. 1997). Recent pharmacological studies showed that berberine also possesses antifungal (Stermitz et al. 2000; Vollekova et al. 2003), anti-inflammatory (Kupeli et al. 2002; Kuo et al. 2004). antioxidative (Rockova et al. 2004), and analgesic (Yesilada and Kupeli 2002) effects. On the other hand, extracts of FE have protective effects on castor oil-induced diarrhea in mice and treating abdominal pain, acid regurgitation, nausea induced by ulcer (Yu et al. 2000). The ethanol extract of FE and its bioactive components (evodiamine and rutaecarpine) also exhibited anti-inflammatory activities which could be in part explained by their different potentials for inhibiting NADPH oxidase-dependent reactive oxygen species and/or inducible nitric oxide synthase-dependent nitric oxide production in activated inflammatory cells (Ko et al. 2007). FE has obviously anti-HP activity (Zuo 2001). Therefore, in ZJW there are multiple anti-ulcers properties of RC and FE. That is to say, although FE stimulates CA secretion induced by ACh, it has other potential targets which are associated with protection for gastric ulcers. These data give the possibility that RC, a component of ZJW, antagonizes the adverse effects of FE on CA secretion and synergistically enhances the therapeutic effects of FE in the treatment of gastric ulcers. To confirm this possibility, further in vivo administration of ZJW, RC and FE to animals or humans will be required in future study.

In summary, we have demonstrated that ZJW, a formula comprised of RC and FE, decreases CA secretion induced by a natural secretagogue ACh via lowering [[[Ca.sup.2+]]sub.i] and that RC antagonizes stimulatory effects of FE on CA secretion in cultured bovine adrenal medullary cells. The present findings may add new pharmacological actions of ZJW in the treatment of gastric ulcer and serve to ensure the safety and effectiveness of TCM formula.

Acknowledgements

This research was supported by the National Natural Science Funds (30672631), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (706013), and the special project of Ministry of Science and Technology in province (2007, BA147B01), the project of Technology Developement Plan of Tianjin (06TXTJJC13600).

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F.-R. Zhao (a), (b), (c) H.-P. Mao(a), (b), (c), H. Zhang (a), (b), (c), (d) L.-M. Hu.(a), (b), (c) H. Wang (a), (b), (c) Y.-F. Wang (a), (b), (c) N. Yanagihara (d), X.-M. Gao(a), (b), (c), *

(a)Key Laboratory of Pharmacology of Traditional Chinese Medical Formulae, Ministry of Education. Tianjin University of Traditional Chinese Medicine. Tianjin 300193. China

(b) Tianjin Key Laboratory of Traditional Chinese, Medicine Pharmacology. Tianjin University of Traditional Chinese Medicine. Tianjin 300193. China

(c) Institute of Traditional Chinese Medicine Research. Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China

(d) Department of Pharmacology. University of Occupational and Environmental Health, School of Medicine. I-I, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

* Corresponding author at: Institute of Traditional Chinese Medicine Research, Tianjin University of Traditional Chinese Medicine, #312 Anshan xi Road, Nankai District, Tianjin 300193. China. Tel.: +86 022 23051027; fax: +86 022 23051066.

E-mail address: gaoxiumei@tjutcm.edu.cn (X.-M. Gao).

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doi:10.1016/j.phymed.2009.10.010

[C]2009 Elsevier GmbH. All rights reserved.
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Author:Zhao, F.-R.; Mao, H.-P.; Zhang, H.; Hu, L.-M.; Wang, H.; Wang, Y.-F.; Yanagihara, N.; Gao, X.-M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jul 1, 2010
Words:5480
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