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Pinoresinol-4,4'-di-O-[beta]-D-glucoside from Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse embryo fibroblasts.

Abstract

Lignans are major constituents of plant extracts and have important pharmacological effects on mammalian cells. Here we showed that pinoresinol-4,4'-di-O-[beta]-D-glucoside (PDG) from Valeriana officinalis induced calcium mobilization and cell migration through the activation of lysophosphatidic acid (LPA) receptor subtypes. Stimulation of mouse embryo fibroblast (MEF) cells with 10 [micro]M PDG resulted in strong stimulation of MEF cell migration and the [EC.sub.50] was about 2[micro]M. Pretreatment with pertussis toxin (PTX), an inhibitor of [G.sub.i] protein, completely blocked PDG-induced cell migration demonstrating that PDG evokes MEF cell migration through the activation of the [G.sub.i]-coupled receptor. Furthermore, pretreatment of MEF cells with Kil6425 (10 [micro]M), which is a selective antagonist for [LPA.sub.1] and [LPA.sub.3] receptors, completely blocked PDG-induced cell migration. Likewise, PDG strongly induced calcium mobilization, which was also blocked by Ki16425 in a dose-dependent manner. Prior occupation of the LPA receptor with LPA itself completely blocked PDG-induced calcium mobilization. Finally, PDG-induced MEF cell migration was attenuated by pretreatment with a phosphatidylinositol 3-kinase (P13K) inhibitor such as LY294002. Cells lacking downstream mediator of PI3K such as Aktl and Akt2 (DKO cells) showed loss of PDG-induced migration. Re-expression of Aktl (but not Akt2) completely restored PDG-induced DKO cell migration. Given these results, we conclude that PDG is a strong inducer of cell migration. We suggest that the pharmacological action of PDG may occur through the activation of an LPA receptor whereby activation of PI3K/Akt signaling pathway mediates PDG-induced MEF cell migration.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Pinoresinol-4,4'-di-O-[beta]-D-glucoside; Valeriana officinalis; Lysophosphatidic acid; Migration; Phosphatidylinositol 3-kinase; Akt

Introduction

The crude extract of Valeriana officinalis root is used for traditional medicine as a mild sedative and tranquilizer in many countries (Houghton, 1999). The genus Valeriana consists of 200 species and belongs to the family of Valerianaceae which is widely distributed throughout the world. Valeriana officinalis has been used as an anticonvulsant, for its hypnotic effects, and as an anxiolytic in some countries (Carlini, 2003). In addition, it has been demonstrated that valerian has mild central nervous system (CNS)-depressant effects in mice (Leuschner et al., 1993) as well as in some clinical studies (Fugh-Berman and Cott, 1999). Valerian also has neuroprotective effect by inhibiting excess calcium influx (Malva et al., 2004). However, the molecular mechanism of the CNS-related action of valerian is still unclear.

Valeriana officinalis consist of over 150-200 chemical constituents including flavonoids and lignans. A flavo-noid such as 6-methylapigenin in Valeriana officinalis has a benzodiazepine binding site (Wasowski et al., 2002) and has sedative as well as sleep-enhancing activity (Marder et al., 2003). Lignans from Valeriana officinalis have antioxidative and vasorelaxant effects (Piccinelli et al., 2004). Structurally, pinoresinol is one of the simplest lignans; it is a dimer of coniferyl alcohol and is frequently present in woody or fibrous plants. Recently, it has been reported that 8-hydroxypinoresinol has affinity for 5-[HT.sub.1A] receptors at low micromolar concentrations (Bodesheim and Hoelzl, 1997). In addition, 4'-O-[beta]-D-glucopyranosyl-9-0-(6"-deoxysaccharosyl)olivil has been demonstrated to have affinity for [A.sub.1] adenosine receptors (Schumacher et al., 2002). Although several reports have demonstrated the ability of pinoresinol modulates several CNS receptors, stimulatory and inhibitory effects of other cell physiology are largely unknown.

Chemotaxis or directional cell migration plays a critical role in tissue development, immune response, and tissue repair. In pathological conditions, aberrant signaling leads to enhanced chemotactic migration such as tumor metastasis (Lauffenburger and Horwitz, 1996). Cell migration is initiated by the activation of cell surface receptors such as growth factor receptors and G protein-coupled receptors (GPCR), leading to the activation of PI3K/Akt signaling pathways, calcium mobilization, and activation of small G proteins.

We aimed to identify novel lignans inducing chemotactic migration. To this end, we screened pure lignan compounds from Valeriana officinalis and found that pinoresinol-4,4'-di-O-[beta]-D-glucoside (PDG) (Fig. 1) strongly induced migration of mouse embryo fibroblast cells. Further in vitro analysis showed that PDG evokes chemotactic migration through the activation of [G.sub.i]-coupled receptors, calcium mobilization, and PI3K/Akt signaling pathways.

[FIGURE 1 OMITTED]

Materials and methods

Plant materials

Powdered Valeriana officinalis (VO) roots were obtained from Frontier Company (2000 Frontier, IA 52318, Norway). Samples were deposited at the University of Mississippi, National Center for Natural Products Research. The finely ground roots of valerian (1 kg) were successively refluxed with hexane, chloroform and methanol overnight. After cooling, the suspension was filtered, and the methanolic solution was evaporated under reduced pressure. The brown, oily residue (10% of original weight) was imbedded in silica.

Extraction and spectral analysis of pinoresinol-4,4'-di-O-[beta]-D-glucoside

The MeOH extract (52 g) was evaporated in vacuo and chromatographed on a silica gel (40 [micro]m, J.T. Baker, NJ, USA) column (70 x 8.0 cm) with a step gradient 5%, 10%, 15% MeOH in chloroform (each 21) to get 15 fractions. Fractions were collected and checked by TLC on silica gel 60 [F.sub.254] and reverse phase (RP) glass plates. Fraction 8 (1444 mg) was separated on a Sephadex 20 column (60 x 3.0 cm) with MeOH to give a pinoresinol-4,4'-di-O-[beta]-D-glucoside (PDG) (234 mg). The chemical structure of PDG was verified by LC-MS (Bruker BioApex FT mass spectrometer) and NMR analysis (Bruker DRX 400 spectrometer). Optical rotations were recorded on a JASCO DIP-370 digital polarimeter. IR spectra were recorded on an AATI Mattson Genesis Series FTIR. NMR spectra ([.sup.1]H, [.sup.13]C) were recorded in [CDC1.sub.3] on a Bruker DRX 400 spectrometer operating at 400MHz for [.sup.1]H and 100 MHz for [.sup.13]C, running gradients, and using residual solvent peaks as internal references. High-resolution mass spectra were recorded on a Bruker BioApex FT mass spectrometer.

Other materials

Dulbecco's modified Eagle's medium (DMEM), trypsin-EDTA, fetal bovine serum (FBS), and antibiotics were purchased from Cambrex Corp. Anti-Aktl/ PKB[alpha] antibody and anti-Akt2/PKB[beta] antibodies were purchased from Upstate Biotech. ChemoTx membrane (8 [micro]m pore size) was purchased from Neuro Probe Inc. Recombinant human platelet-derived growth factor-BB (PDGF-BB) and all other high-quality reagents were obtained from Sigma-Aldrich unless indicated elsewhere.

Establishment of mouse embryo fibroblast (MEF) cells

MEF cells were isolated the same way as described previously (Yun et al., 2008). Embryos were dissected from pregnant Aktl/PKB[[alpha].sup.[+/-]]; Akt2/PKB[[beta].sup.[+/-]] females that had been bred to Aktl/PKB[[alpha].sup.[+/-]]; Akt2/PK[[beta].sup.[+/-]] males. The yolk sacs, heads, and internal organs were isolated and used for genotyping by RT-PCR. Carcasses were treated with trypsin-EDTA for 30 min at 37 [degrees]C, and clumps of cells were disrupted by chopping with scissors. After centrifugation, the cells were re-suspended in culture medium (DMEM supplemented with 10% FBS and antibiotics) and maintained at 37 [degrees]C in 5% [CO.sub.2]. Primary cells were immortalized by continuous culturing for 30 passages.

Retroviral infection

Generation of viral supernatant was done as previously reported (Yun et al., 2008). Briefly, ecotropic BOSC23 cells were transiently transfected with pVSV/G and pGag/pol, pantropic retroviral packaging constructs, and retroviral vector containing Aktl/PKB[alpha], Akt2/PKB[beta]. Cell-free viral supernatants were mixed with one volume of complete medium in the presence of 8 [micro]g/ml polybrene and used to infect immortalized MEF cells. Cells expressing Akt/PKB are sorted by flow cytometer (BD Biosciences) and used for the experiments.

Western blotting

Cells were lysed in 20 mM Tris-HCl, pH 7.3, 1 mM EGTA/EDTA, 1% Triton X-100, 1 mM [Na.sub.3][VO.sub.4], 10 [micro]g/ml leupeptin, and 10 [micro]g/ml aprotinin. After centrifugation at 12,000 rpm, 30 [micro]g of total protein was loaded into 10% polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were incubated with indicated primary antibody and HRP-conjugated secondary antibody. Protein bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotechnology).

Cell migration assay

MEF cells were grown and serum starved for 6h before plating on the ChemoTx membrane. Cells were detached with trypsin-EDTA and washed with serum-free DMEM. For the migration assay, the bottom side of the ChemoTx membrane was coated with type I collagen for 30 min, and 2 x [10.sup.4] serum-starved cells (in 50 [micro]l) were placed on the top side of the ChemoTx membrane. Migration was induced by submerging the ChemoTx membrane in serum-free medium either in the presence or in the absence of chemotactic attractants for 3 h. The ChemoTx membrane was fixed with 4% paraformaldehyde, and non-migratory cells on the top side of the membrane were removed by gently wiping with a cotton swab. The membrane was stained with DAPI, and migrating cells were counted under the fluorescence microscope at 10 x magnification (Axiovert 200).

Measurement of intracellular calcium concentration

Intracellular calcium concentration was measured using fura-2/AM, a calcium-sensitive fluorescent dye, as described previously (Grynkiewicz et al., 1985). Briefly, a total of 2 x [10.sup.6] MEF cells were incubated with 3 [micro]M fura-2/AM at 37 [degrees]C in fresh serum-free DMEM medium and stirring for 50min. Cells (2 x [10.sup.5]) were aliquoted for each assay into Locke's solution (154 mM NaCl, 5.6 mM KC1, 1.2 mM [MgCl.sub.2], 5 mM HEPES, pH 7.3, 10 mM glucose, 2.2 mM [CaCl.sub.2], and 0.2 mM EGTA). Fluorescence was measured at 500 nm at excitation wavelengths of 340 and 380 nm.

Statistical analysis

Results are expressed as the mean[+ or -]S.D. of two independent experiments (n = 3 for each experiment). When comparing the two groups, an unpaired Student's t-test was used to address differences. P-values less than 0.05 were considered significant.

Results

Isolation of pinoresinol-4,4'-di-O-[beta]-D-glucoside (PDG) and migratory effect on mouse embryo fibroblast cells

Pinoresinol-4,4'-di-O-[beta]-D-glucoside (Fig. 1) was originally isolated from Eucommia ulmoides and Valeriana officinalis (Schumacher et al., 2002; Sih et al., 1976). Briefly, pinoresinol-4,4'-di-O-[beta]-D-glucoside was obtained as colorless needles (from MeOH) [[alpha]] (26), (1) d -19.01 (13.64 (c 0.59, MeOH)). The positive HRTOFMS indicated a molecular ion at m/z 681.2202 corresponding to [[M-H].sup.-], thus indicating a molecular formula of [C.sub.32][H.sub.42][O.sub.16]. The IR spectrum displayed a band at 2937, 1605, 1524 [cm.sup.-1]. The [.sup.13]C NMR and DEPT spectra showed 16 signals (2 x) with symmetric compound, including 12 carbons for two aromatic rings, two methylenes (8 71.6 (2 x), C-9 and C-9'), two methines ([delta] 54.3 (2 x) C-7 and C-7), two methines ([delta] 85.9, C-8 and C-8'), two methoxy carbons ([delta] 55.6 (2 x )), and six carbon resonances corresponding to a sugar moiety.

The [.sup.1]H NMR spectrum showed two ABX type coupling patterns, thus indicating the presence of two 1,3,4-trisubstituted phenyl groups [6.99 (2H), d, J= 1.2 Hz, H-2 and H-2'), 7.12 (2H, d, J= 8.4 Hz, H-5 and H-5'), and 6.88 (2H, dd, J= 8.8 and 1.6 Hz, H-6 and H-6'), two benzylic oxymethine protons at 4.71 (2H, s, H-7 and H-7'), two methylenes bearing an oxygen function at 5 4.20 (2H, d, J = 7.2 Hz, H-9a and H-9'a), 3.84 (2H, d, J=7.2 Hz, H-9b, H-9'b), two O-methyl singlets at [delta] 3.83 (6H, s), two anomeric protons at 8 4.81 (2H, d, J = 7.2 Hz), and signals in the [delta] 3.39-4.81 region attributable to a sugar moiety. Assignment of all [.sup.1]H and [.sup.13]C NMR signals were based on HMBC, HSQC, and DQFCOSY experiments. The COSY spectrum confirmed the presence of two 1,3,4-trisubstituted phenyl groups showing connectivities between the hydrogens H-6, H-2, and H-5 of ring A and the hydrogens H-6', H-2', and H-5' of ring B. The aromatic carbon shifts suggested that the aryl group is linked to a glycosyl moiety. These spectroscopic data suggested that pinor-esinol 4,4'-di-O-[beta]-D-glucoside (PDG) is a glycosyl derivate of pinoresinol (Fig. 1A). Stimulation of MEF cells with PDG significantly enhanced migration ([EC.sub.50]~2[micro]M) and the effect was saturated at 5 [micro]M (Fig. 2B). In addition, other pinoresinol derivatives including pinoresinoi-4-O-[beta]-D-glucoside, 8-hydroxypi-noresinol-4'-O-[beta]-D-glucoside, berchemol-4'-O-[beta]-D-glu-coside, massoniresinol-4'-O-[beta]-D-glucoside, and trans-coniferin showed similar effect on the MEF cell migration. However, glucopyranoside and adenosine did not affect MEF cell migration (data not shown).

[FIGURE 2 OMITTED]

PDG-induced MEF cell migration was mediated by [G.sub.i]coupled receptors

Since we have previously reported that both growth factor receptors and G protein-coupled receptors are involved in the regulation process of MEF cell migration (Kim et al., 2008a, 2008b), we next evaluated the responsible receptor type during PDG-induced MEF cell migration. As shown in Fig. 2A, pretreatment of cells with a receptor tyrosine kinase inhibitor such as Genistein (10 [micro]M) completely abolished PDGF (50ng/ml)-induced MEF cell migration (p<0.05), whereas Genistein did not affect LPA- or PDG-induced MEF cell migration (p>0.05). In contrast, PDG- or LPA-induced MEF cell migration was completely blocked by pretreatment of cells with a [G.sub.i] inhibitor such as pertussis toxin (PTX) demonstrating that PDG activates [G.sub.i]-coupled receptor and induces MEF cell migration. To further confirm the mechanism of PDG action during the MEF cell migration, we pretreated Ki16425, which is an LPA receptor antagonist. As shown in Fig. 2B, Ki16425 completely blocked PDG-induced MEF cell migration (p<0.05) as well as LPA-induced MEF cell migration. However, PDGF-induced MEF cell migration was not affected by Ki16425 pretreatment (p > 0.05).

PDG-induced calcium mobilization through the activation of LPA receptors

Previous reports have shown that a [G.sub.i]coupled receptor strongly evokes calcium mobilization (Lee et al., 2007). In addition, our results have shown that PDG activates [G.sub.i]coupled receptors during cell migration. Therefore, we next examined the effect of PDG on calcium mobilization. As shown in Fig. 3A, stimulation of cells with [G.sub.i]-coupled receptor agonists such as LPA and sphingosine-1 -phosphate (SIP) strongly induced calcium mobilization. Likewise, stimulation of cells with PDG evoked calcium mobilization about 50% of maximum LPA-stimulated calcium release. Since previous results indicated that PDG-dependent MEF cell migration was sensitive to an LPA receptor antagonist (Ki16425), we investigated the effect of LPA receptor pre-occupation on the PDG-induced calcium mobilization. As shown in Fig. 3B, prior occupation of an LPA receptor by pretreatment of LPA completely blocked PDG-induced calcium mobilization. Likewise pretreatment of PDG strongly attenuated LPA-induced calcium mobilization, whereas SIP-induced calcium mobilization was not affected by PDG pretreatment. Finally, treatment of cells with Kil6425 (10 [micro]M), known as an [LPA.su1] and [LPA.sub.3] receptor selective antagonist, completely blocked PDG-induced calcium mobilization and partially blocked LPA-induced calcium mobilization (Fig. 3C).

[FIGURE 3 OMITTED]

PDG-induced MEF cell migration was mediated by PI3K/Akt signaling pathways

Since LPA-induced MEF cell migration was mediated by PI3K/Akt signaling pathways (Kim et al., 2008b) and PDG-induced calcium mobilization was mediated by LPA receptor signaling (Fig. 3), we next investigated the role of PI3K/Akt signaling pathways in PDG-induced MEF cell migration. As shown in Fig. 4A, PDG-induced MEF cell migration was completely blocked by pretreatment of LY294002 (10 [micro]M), which is a PI3K inhibitor; however, inhibition of ERK and p38 MAPK pathways did not affect PDG-induced MEF cell migration (p > 0.05). To delineate the signaling pathway downstream of PI3K, we examined PDG-induced migration in cells lacking Akt1 and/or Akt2. As shown in Fig. 4B cells lacking Akt1 (1KO and DKO) lost their response to PDG-induced migration, whereas the loss of Akt2 (2KO) did not significantly affect it. Finally, either Akt1 or Akt2 was re-introduced into Akt1 and Akt2 double knock-out (DKO) cells (Fig. 4). Ectopic expression of Akt1 or Akt2 was about 100-fold higher than endogenous Akt1 and Akt2 in wild-type cells. DKO cells completely restored PDG-induced migration by re-expression of Akt1 but not by Akt2 (Fig. 4D).

[FIGURE 4 OMITTED]

Discussion

Plant extracts have a variety of physiological effects and have been used in traditional medicine since ancient times. This is especially true for the crude extract of Valeriana officinalis which has CNS-depressant and neuroprotective effects. Lignans are major constituents of crude plant extracts and pinoresinol is structurally one of the simplest lignans. Especially, Eucommia ulmoides extract has been used for antihypertensive, anti-inflammatory, and sedative activity in which lignans are major responsible components mediating those effects. Recent experiments have demonstrated that lignans have binding affinity for serotonin and adenosine receptors (Bodesheim and Hoelzl, 1997; Schumacher et al., 2002). However, the exact biological function and molecular mechanism of pinoresinol in mediating cell migration and calcium mobilization, which are important process for neuronal activity, cardiovascular function, and inflammation, are unknown. In the present study, we provide novel insight that pinoresinol-4,4'-di-O-[beta]-D-glucoside (PDG) is a strong modulator for cell migration and calcium mobilization.

Virtually, we have screened 45 compounds from Valeriana officinalis and Schisandra chinensis for cell migration and vasorelaxation. Gomisin A from Schisandra chinensis was identified as vasorelaxant (Park et al., 2007). Although it has been reported that lignans from Valeriana prionophylla have vasorelaxant activity at high molar concentration (10-100 [micro]M), low molar concentration of PDG (5 [micro]M) did not have vasorelaxant activity (data not shown). However, PDG strongly stimulated cell migration and calcium mobilization at 5 [micro]M, indicating that migration and calcium mobilization rather than vasorelaxation are more susceptible to PDG stimulation. Therefore, we provide novel physiological function of PDG as a strong modulator of cell migration and calcium mobilization, which are important for the inflammation and neural function.

Several lines of evidences support that PDG-depen-dent MEF cell migration is mediated by LPA receptor subtype. First, the possible involvement of receptor tyrosine kinase could be excluded since PDG-dependent cell migration was not affected by pretreatment of receptor tyrosine kinase inhibitor (Fig. 2A). However, PDGF-induced cell migration was completely blocked by receptor tyrosine kinase inhibitor. Second, PDG-induced cell migration was blocked by inactivation of a [G.sub.i]-coupled receptor using pertussis toxin (Fig. 2A), demonstrating that a PDG receptor is [G.sub.i]-coupled. Since LPA receptor is also coupled to [G.sub.i] protein (Hilal-Dandan et al., 2004), LPA-induced cell migration was completely blocked by pretreatment of pertussis toxin (Fig. 2A). Third, it has been shown that Ki16425 is a selective inhibitor of [LPA.sub.1] and [LPA.sub.3] (Ohta et al., 2003), and Ki16425 blocked cell migration induced by both PDG and LPA (Fig. 2B). Therefore, it is possible that PDG-dependent cell migration may be mediated by the activation of an LPA receptor subtype at least in part.

Calcium mobilization is a major downstream response of LPA receptor activation (Lee et al., 2007). In correlation with this, stimulation of MEF cells with LPA strongly evoked calcium release (Fig. 3A). PDG also induced calcium mobilization, although the maximum response was about 50% of LPA-induced calcium mobilization. This may be due to the subtype-selective activation of LPA receptor. For example, inactivation of [LPA.sub.1] and [LPA.sub.3] by high concentration of Ki16425 (10 [micro]M) resulted in complete blocking of PDG-induced calcium mobilization, whereas LPA-induced calcium mobilization was partly blocked at the same concentration of Ki16425 (Fig. 3C). These results demonstrate that PDG may selectively induce [LPA.sub.1] and [LPA.sub.3] to mobilize intracellular calcium. The involvement of an LPA receptor during PDG-induced calcium mobilization was also confirmed by a pre-occupation experiment. For instance, pre-occupation of an LPA receptor by LPA itself completely blocked PDG-induced calcium mobilization and vice versa (Fig. 3B). It is also noteworthy that pretreatment of PDG resulted in partial blocking of LPA-induced calcium mobilization. This may be due to the restricted occupation of [LPA.sub.1] and [LPA.sub.3] receptors. Therefore, PDG may selectively exert its effect on [LPA.sub.1] and [LPA.sub.3] receptor subtype and induces calcium mobilization as well as cell migration.

Previously, we have shown that LPA induces cell migration through the selective activation of PI3K/Aktl signaling pathway (Kim et al., 2008b). For example, cells lacking Aktl (but not Akt2) did not stimulate LPA-dependent migration. LPA-induced DKO cell migration was restored by re-expression of Aktl but not Akt2. Likewise, PDG-induced cell migration was mediated by PI3K and abolished in cells lacking Aktl (Fig. 4). Furthermore, DKO cells restored PDG-induced migration after re-expression of Aktl but not Akt2. These results indicate that PDG-induced cell migration is similar to LPA-induced cell migration. Taken together, we have shown that PDG induces cell migration and calcium mobilization similar to LPA receptor-dependent pathways.

Although it is unequivocal that PDG-induced cell migration and calcium mobilization is partly mediated by the activation of LPA receptor subtype, it is still ambiguous how PDG affects neurological function. Nonetheless, it is possible that PDG-induced activation of LPA receptor may affect neurological function thereby inducing sedative and anxiolytic function. Recent evidences also support the idea that LPA modulates neurological function. For example, neuropathic pain is mediated by the generation of LPA (Inoue et al., 2008). More direct evidence that LPA is involved in the neurological function was discovered by the evaluation of LPA receptor knock-out mice. Mice lacking [LPA.sub.1] receptor showed schizophrenia phenotype in which serotonin efflux was dramatically decreased (Roberts et al., 2005). Serotonergic neuronal activity is modulated by NMD A receptor (Gartside et al., 2007), and administration of NMDA receptor antagonist frequently evokes schizophrenia phenotype (Enomoto et al., 2007). Therefore, both LPA receptor and NMDA receptor modulate serotonergic neuronal activity and serotonin efflux. Since serotonin agonists are frequently used for anxiolytic activity (New, 1990), it is possible that PDG-dependent modulation of LPA receptor activity may contribute to the mechanism of anxiolytic activity of PDG. Evaluation of PDG-dependent behavioral function of mice lacking LPA receptor will provide more insight into this proposed mechanism.

In conclusion, we have defined the novel molecular mechanism of PDG action in cell migration and calcium mobilization. We have provided evidence that PDG could be a novel ligand for [LPA.sub.1] and [LPA.sub.3] receptors. Studies on the underlying mechanism of PDG on LPA-related psychiatric function may shed more light on the evaluation of PDG as an anxiolytic.

Acknowledgements

This study was supported by Technology Development Program for Agriculture and Forestry (106048031SB010), Ministry of Agriculture and Forestry, Republic of Korea, and MRC program of MOST/KOSEF (R13-2005-009) (to S.S.B.).

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Kee Hun Do (a), (1), Young Whan Choi (b), (1), Eun Kyoung Kim (a), Sung Ji Yun (a), Min Sung Kim (a), Sun Young Lee (a), Jung Min Ha (a), Jae Ho Kim (c), Chi Dae Kim (a), Beung Gu Son (b), Jum Soon Kang (b), Ikhlas A. Khan (d), Sun Sik Bae (a), *

(a) Department of Pharmacology and MRC for Ischemic Tissue Regeneration and Medical Research Institute, School of Medicine, Pusan National University, Busan 602-739, Republic of Korea

(b) College of Natural Resources & Life Sciences, Pusan National University, Gyungnam 627-706, Republic of Korea

(c) Department of Physiology and MRC for Ischemic Tissue Regeneration and Medical Research Institute, School of Medicine, Pusan National University, Busan 602-739, Republic of Korea

(d) National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA

* Corresponding author. Tel.: 4-82 51 240 7944; fax: + 82 51 244 1036.

E-mail address: sunsik@pnsan.ac.kr (S.S. Bae).

(1) Equally contributed to this work.

doi: 10.1016/j.phymed.2008.12.002
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Author:Do, Kee Hun; Choi, Young Whan; Kim, Eun Kyoung; Yun, Sung Ji; Kim, Min Sung; Lee, Sun Young; Ha, Jun
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9SOUT
Date:Jun 1, 2009
Words:4732
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