Ginsenoside [F.sub.2] induces apoptosis in humor gastric carcinoma cells through reactive oxygen species-mitochondria pathway and modulation of ASK-1/JNK signaling cascade in vitro and in vivo.
Ginsenoside [F.sub.2]([F.sub.2]) is a potential bioactive metabolite of major ginsenosides. The potential anti-cancer effect of [F.sub.2] in gastric cancer cells has not been appraised. This study investigated the effects of [F.sub.2] on the production of reactive oxygen species (ROS). We also investigated the in vitro and in vivo effects of [F.sub.2] on the downstream signaling pathways leading to apoptosis in human gastric cancer cells. The in vitro data revealed that [F.sub.2] induces ROS accumulation followed by a decrease in mitochondrial transmembrane potential (MTP), and the release of cytochrome c (cyto c), which induced the caspase-dependent apoptosis. Further assay indicated that modulation of ASK-l/JNK pathway contributes to apoptosis. In vivo, [F.sub.2] exhibits the obvious anti-cancer effect compared with cisplatin with no obvious toxicity. Jointly, these results suggest that [F.sub.2] induces apoptosis by causing an accumulation of ROS and activating the ASK-1 /JNK signaling pathway. This provides further support for the use of [F.sub.2] as a novel anticancer therapeutic candidate.
Subcutaneous tumor model
Gastric cancer is the fourth most commonly diagnosed cancer and the second most common cause of cancer-related deaths worldwide (Garcia et al., 2007). Five-year survival was 36% in patients with operable disease who were assigned to perioperative chemotherapy (Cunningham et al., 2006).
In recent years, the exploration of protopanaxadiol (PPD) type ginsenosides for new anti-carcinogenic agent is of great interest. Structural-function studies of ginsenosides in inducing apoptosis, reducing cell proliferation showed that the antitumor effects are related to some structural feathers. Popovich et al. have found that presence of sugars in protopanaxadiol aglycone structures reduces the potency to induce apoptosis (Popovich and Kitts, 2002). Qi et al. reviewed that the anticancer effect of ginsenosides increases with the decrease of sugar number (Qi et al., 2010). Our recent published paper showed that ginsenosides with four or more sugar molecules (e.g., [Rb.sub.1] and Rc) show no significant antitumor effects (Mao et al., 2012). In addition, sugar moiety at C-6 significantly reduces the anticancer activities of ginsenosides (Li et al., 2009). Stereoselective interactions with lipid membranes may also affect the antiproliferative activity by 20(R)- and 20(S)-ginsenosides. It has been found that 20(S)-[Rg.sub.3],20(S)-[Rh.sub.2] and 20(S)-PPD show stronger chemopreventive effects than their 20(R)-sterioisomers (Lee et al., 2009; Li et al., 2009; Wang et al., 2007). In light of these structuralactivity relationships, it was inferred that 20(S)-ginsenoside [F.sub.2] ([F.sub.2]), a PPD type ginsenoside with one sugar molecular at C-3 and one sugar molecule at C-20 (structure see Fig. 1), may have potential antitumor effects. But information is very limited on the bioactivity of [F.sub.2] (Mai et al., 2012; Shin et al., 2012). And the anticancerous effects and detailed molecular mechanism in gastric cancer cell lines remains unclear.
Apoptosis is characterized by a series of distinct biochemical and morphological changes, including increase in reactive oxygen species (ROS) level, activation of caspases, cell shrinkage, chromatin condensation. (Matthew et al., 1997; Simon et al., 2000; Wyllie, 1995). One of the most significant events in apoptosis is mitochondrial dysfunction. Loss of mitochondrial transmembrane potential (MTP) elicits the release of cytochrome c (cyto c) from mitochondria to cytosol, thereby activating the caspase-cascade system (Green and Reed, 1998; Thornberry and Lazebnik, 1998). Moreover, it is evident that the accumulation of ROS is required for the activation of apoptosis signal-regulated kinase-1 (ASK-1)/c-Jun N-terminal protein kinase (JNK) pathway (Hassan et al., 2006; Kunz et al., 2006). But the mechanisms by which the apoptotic effects of ginsenosides such as [F.sub.2] may have on this pathway remain a mystery.
With great potential in anti-carcinogenic activity and little study about the effect of [F.sub.2] in gastric carcinoma cell, we proved that [F.sub.2] could induce the accumulation of ROS production and a decrease in MTP, by triggering ASK-1/JNK apoptosis pathway (Fig. 2). Furthermore, in vivo data also showed [F.sub.2] has potential anti-cancer activity because it inhibits the growth of human gastric adenocarcinoma SGC790 cells.
Materials and methods
Reagents and antibodies
Ginsenoside [F.sub.2] was isolated previously from leaves of P. notoginseng by a series of chromatographic procedures. [F.sub.2] has a molecular mass of 784 Da and was isolated with 98% purity. Cisplatin (DDP) of the highest analytical grade was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies of ASK-1, JNK, cyto c, caspase 3, bcl-2, PARP together with all secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The caspase 9 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell lines and cell culture
SGC7901 cells were purchased from American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), 100 [micro]g/ml streptomycin, 100 [micro]g/ml penicillin and grown at 37[degrees]C in 5% carbon dioxide.
MTT proliferation assay
The effects of [F.sub.2] on SGC-7901 cells were investigated by MTT (methylthiazolely diphenyl-tetrazolium bromide) assays. The tumor cells (2 x [10.sup.4]/well in 180 [micro]l) were seeded into 96-well plates for 24 h before drug treatment. DDP, exhibiting activity against a variety of cancer cells (Park et al., 2002; Henkels and Turchi, 1999; Blanc et al., 2000), was used as positive control. After treatment with various concentrations of [F.sub.2] and DDP for 72 h, the cell plates were treated with 20 [micro]l of MTT solution (5 mg/ml in PBS) for an additional 4h at 37[degrees]C. After treatment, the formazan crystals in viable cells were solubilized with 120 [micro]l DMSO and the absorbance in each well was read at 570 nm using a Safire 2 microplate reader (Tecan, Switzerland). The cell survival percentages were calculated by dividing the mean OD of compound-containing wells by that of control wells.
FACS analysis was performed to detect apoptosis. Briefly, 5 x [10.sup.5] cells were seeded in 6-well plates for 24 h, and then exposed to various treatments for additional 48 h. All the cells including the floating cells were collected by centrifugation at 1000 x g for 10 min according to the manufacturer's instructions. Cell apoptosis was analyzed using a FITC-Annexin V Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA).
Western blot analysis
After the treatment of the indicated concentration of [F.sub.2] (40, 80 and 100 [micro]M) for 24 h, cells were harvested, washed with cold PBS (pH 7.4) and lysed with ice-cold lysis buffer (50 [micro]M Tris-HCl, 150 [micro]M NaCl, 1 [micro]M EGTA, 1 [micro]M EDTA, 20 [micro]M NaF, 100 [micro]M [Na.sub.3]V[0.sub.4], 1% NP40,1 [micro]M PMSF, 10 [micro]g/ml aprotinin and 10 [micro]g/ml leupeptin, pH 7.4) for 30 min and centrifuged at 12,000 x g for 30 min at 4[degrees]C. The protein concentration of the clear supernatant was quantified using Bio-Rad Protein Assay Kit.
Approximately 30|xg of protein was loaded into a 10-15% sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE). Thereafter, proteins were electrophoretically transferred to nitro-cellulose membrane and non-specific sites were blocked with 5% skimmed milk in 1% Tween-20 (Sigma-Aldrich) in 20 [micro]M TBS (pH 7.5) and reacted with a primary polyclonal antibody, ASK-1, JNK, bcl-2, cyto c, caspase 9, caspase 3, PARP and [beta]-actin for 4h at room temperature. After washing with TBS three times (5 min each), the membrane was then incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody. The signal was observed and developed with Kodak film by exposure to Enhanced Chemiluminescence (ECL) plus Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ, USA).
Mitochondrial transmembrane potential ([DELTA][[PSI].sub.m]) assessment
Mitochondrial transmembrane potential ([DELTA][[PSI].sub.m]) was measured by using a Mitochondrial Membrane Sensor Kit containing JC-1, a sensitive fluorescent dye, as described by the manufacturer (BD Bioscience, CA). Briefly, after treatment with different concentrations of [F.sub.2] (40,80 and 100 [micro]M) for 24 h, the SGC7901 cell were harvested with ice-cold PBS and were resuspended in RPMI-1640 medium at a density of 5 x [10.sup.5] cells/ml. And then incubated with 10 [micro]M JC-1 for 15 min at 37[degrees]C in the dark and observed under a fluorescence microscope (Olympus 1X51, Japan). Red fluorescence is attributable to a potential-dependent aggregation in the mitochondria. Green fluorescence, reflecting the monomeric form of JC-1, appeared in the cytosol after mitochondrial membrane depolarization.
Measurement of ROS formation
Generation of ROS was assessed by using the fluorescent signal H2DCFDA (2,7-dichlorodihydrofluorescein), a cell-permeable indicator for ROS initially shown to react with [H.sub.2][0.sub.2] (Bobyleva et al., 1998). As described previously, H2DCFDA was oxidized to a highly green fluorescent DCF (2,7-dichlorofluorescein) by the generation of ROS. The SGC7901 cells were pretreated with 40, 80 and 100 [micro]M for 24 h. The cells were incubated with 100 [micro]M/l [H.sub.2]DCFDA in PBS for 30 min. After 30 min at 37[degrees]C, DCF fluorescence (excitation, 485 nm and emission, 525 nm) was observed under a fluorescent microscope (Olympus 1X51, Japan). The fluorescence intensity was measured using a fluorescence plate reader (BD Falcon, CA) at Ex./Em. = 488/525 nm.
Subcutaneous tumor model of gastric cancer
Six week old female athymic nude mice (BALB/c, nu/nu) were purchased from Shanghai Slac Animal Center (Shanghai, China). All BALB/c athymic nude mice (Shanghai Slac Animal Center, Shanghai, China) were maintained under specific pathogen-free conditions, provided with sterilized food and water, and housed in positive pressure isolators with 12 h light/dark cycles. Aliquots of SGC7901 cells (2 x [10.sup.6] cells/0.1 ml) were injected subcutaneously into the left dorsal area of the mice. The tumor growth and body weight of the mice were monitored every other day. When the diameter of the tumor reached 5 mm, tumor-bearing mice were randomly divided into treatment and control groups (n = 5 mouse/group). Ginsenoside [F.sub.2] was administered via intragastric (i.g.) administration at a dose of 0.8 and 1.6 mg/kg everyday; DDP was administered via intraperitoneal (i.p.) injection at a dose of 2 mg/kg/week. The control group received PBS only. Mice were exposed for 18 days. The volume of the tumor was calculated from the formula V= 1/2 (A x [B.sup.2]), where A indicates the length and B indicates the width of the tumor measured by calipers. After treatment, all the mice were sacrificed and the tumors were excised from the body for analysis. All the experiments were carried out in accordance with the Guidelines for Animal Experimentation of China Pharmaceutical University (Nanjing, China) and the protocols had been approved by the Animal Ethics Committee of this institution.
Immunohistochemical analysis and TUNEL
4 [micro]m sections from formalin-fixed paraffin embedded xenograft tissue were placed onto poly-L-lysine coated glass slides and dried for 1 h at 60[degrees]C before being deparaffinised in xylenes and then rehydrated through a graded alcohol series. 10 [micro]M citric acid buffer, pH 6.0 was used in a standard microwave-based antigen retrieval procedure. Sections were microwaved in a pressure vessel for 15 min before being immunostained on a DAKO autostainer using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's protocol. Briefly, sections were blocked in diluted normal blocking serum for 20 min followed by 1 h incubation with primary rabbit polyclonal antibody ASK-1, JNK, cyto c, caspase 3, bcl-2, PARP (Cell Signaling Technology, Danvers, MA, USA), caspase 9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and TUNEL (Roche Diagnostics, Laval, Q.C, Canada). Sections were then incubated with biotinylated secondary antibody for 30 min followed by Vectastain (Elite ABC reagent) for another 30min. Liquid diaminobenzidine (DAB) (DAKO, Carpinteria, CA, USA) was used as a chromogenic agent for 5 min and sections were counterstained with Mayer's hematoxylin.
Results were expressed as mean [+ or -] standard error. These data presented here were obtained in at least three independent experiments. The differences between the groups were examined for statistical significance using the Student's t-test with SPSS software.
[F.sub.2] shows potent cytotoxicity and induces apoptosis in SGC7901 cells
MTT assays were performed to discern if [F.sub.2] causes a decrease in the viability of SGC7901 cells. As shown in Fig. 3A, following 24 h treatment, [F.sub.2] obviously inhibited the viability of SGC7901 cells and the IC50 value was 49.9 [+ or -] 4.2 pM. The substantial morphological changes observed in [F.sub.2] treated SGC7901 cells for 24 h were examined and photographed by the inverted light microscope (Fig. 3B). Damaged cells became round and shrunken, while the untreated cells retained the normal size and shape. In case of SGC7901 cell for [F.sub.2], [IC.sub.50] value was >110 [micro]M at 48 h of treatment (data not shown). Treatment with <40 [micro]M concentration of [F.sub.2] did not produce any statistically significant reduction in cell proliferation. Based on the observed amounts of inhibition in SGC7901 cells, we have selected 0,40, 80, 100 [micro]M doses for our further studies. We hypothesized that the decrease in cell viability caused by [F.sub.2] in SGC7901 cells might be mediated through the initiation of apoptotic cascade. To confirm the assumption, the cells were treated with different concentrations of [F.sub.2] (0,40,80, 100 [micro]M) for 24 h and analyzed for apoptosis using Annexin V/PI double-staining method. Compared with the vehicle-treated controls (0 [micro]M), the early and median apoptotic cells (low right section of fluorocytogram) were increased (from 2.7% to 37.0%, respectively) and the necrotic cells (upper right section of fluorocytogram) also increased strikingly (from 10.6% to 57.4%) after treated with 40, 80 and 100 [micro]M [F.sub.2] for 24 h (Fig. 4A and B). The results indicated that [F.sub.2] can effectively induce cell growth inhibition and apoptosis, and has potent cytotoxicity in human gastric cancer cells in vitro.
[F.sub.2] decreases MTP and increases ROS levels in SGC7901 cells
MTP change in mitochondria is a crucial stage in apoptosis induced by drugs (Kroemer et al., 2007). By staining tumor cells SGC7901 with a fluorescent probe JC-1, we detected the loss of MTP after the treatment of cells with [F.sub.2]. Red fluorescence of JC-1 dimers was present in areas with high MTP, while green fluorescence of JC-monomers was prevalent in areas with low MTP. Fig. 5A shows a remarkable increase in green fluorescence of JC-1 monomers in [F.sub.2] treated cells, indicating a reduction in MTP.
Mitochondria are the major source of ROS production (Kroemer et al., 2007; Nigam et al., 2009), so changes in ROS level after [F.sub.2] treatment are shown in Fig. 5B. Gradual increase of DCF fluorescence in SGC7901 cells with increasing concentrations of [F.sub.2] was observed after the treatment, suggesting excessive ROS was accumulated.
[F.sub.2] triggers apoptotic pathway
Mitochondrial dysfunction associated with ROS-mediated cell death induces the production of pro-apoptotic proteins such as cyto c. Proteins like cyto c subsequently cause caspase-dependent or caspase-independent apoptosis when they are released from the mitochondria into the cytosol (Kroemer et al., 2007; Roue et al., 2003). Thus, as shown in Fig. 6A, we measured the activation of caspases family proteins, such as caspase 9 and caspase 3 by Western blotting. It is well known that the activation of caspase 3 during apoptosis can cause the cleavage of PARP, a major indicator of apoptosis. So we further determined the expression of PARP. Our results showed that [F.sub.2] dose-dependently decreased the expression of PARP, which supports the theory of an [F.sub.2]-induced apoptosis in SGC7901 cells. Cyto c release is a central step in the apoptosis induced by many death stimuli. Bcl-2 plays a critical role in controlling this step (An et al., 2004). Apoptosis signal-regulated kinase-1 (ASK-1) is a redox-sensitive, mitogen-activated protein kinase that is known to induce c-Jun N-terminal protein kinase (JNK)-mediated inflammation and apoptosis. And JNK can phosphorylates Bcl-2 (Makena et al., 2012). In order to elucidate the upstream [F.sub.2]-induced apoptosis pathways, the protein expression of ASK-1, JNK, Bcl-2, Cyto c were investigated after treatment with the different concentrations of [F.sub.2] for 24 h. Fig. 6B showed a significant decrease in the level of Bcl-2, as well as notable increase of in the levels of ASK-1, JNK, cyto c. At the high concentration of (100 [micro]M) [F.sub.2]i the level of Bcl-2 and PARP decreased by 32.9% and 55.1%, respectively. The protein expressions of ASK-1, JNK, Cyto c, caspase 9, caspase 3 increased about 2.33,1.17,1.65,2.83 and 1.78 fold, respectively.
The anti-tumor effects of [F.sub.2] in vivo
Given that [F.sub.2] demonstrates anti-tumor effects in vitro, we further determined the effects of [F.sub.2] in the human gastric adenocarcinoma cancer xenograft mouse model (Fig. 7A) as described in Section Materials and methods. The illustration of excised tumors of each group was shown in Fig. 7B. As shown in Fig. 7C, tumor growth was much slower for the 1.6 mg/kg [F.sub.2] treated group as compared to the control group. The i.g. administration of [F.sub.2] at a dose of 0.8 mg/kg resulted in no significant tumor growth inhibition. The treatment with [F.sub.2] at a dose of 1.6 mg/kg significantly decreased the tumor growth by 47.14% compared with control group. And the relative tumor volume showed no obvious difference between the 1.6 mg/kg group and the positive control group. In addition, the total tumor weight in [F.sub.2] treated mice at a dose of 1.6 mg/kg was 2.60 [+ or -] 0.2 g as compared to 2.00 [+ or -] 0.88 g in positive control group (p<0.05) (Fig. 7D). We evaluated the changes in body weight during the therapy in four groups. From Fig. 7E, no significant addition of toxicity in terms of progressive weight loss was observed in the [F.sub.2] treated group as compared with the DDP group.
We further tested the apoptotic effects on each group using TUNEL as an indicator of apoptosis. In accordance with the results in vitro, the [F.sub.2] treated group displayed more TUNEL positive cells than did the control group (Fig. 8). To confirm the results of the proteins expression in vitro, immunohistopathological analysis was performed for exploring whether ASK-1, JNK, Bcl-2, cyto c, caspase 9, caspase 3 and PARP could express in vivo. It was indicated that tumors of 1.6mg/kg [F.sub.2] treated group showed increased in ASK-1, JNK, cyto c, caspase 9 and caspase 3 positive cells compared with control group. While Bcl-2 and PARP positive cells decreased in [F.sub.2] treated group (Fig. 9). The results above indicate that [F.sub.2] can induce apoptosis in the subcutaneous tumor model just observed in vitro.
The antitumor activities of ginsenosides from Panax species have attracted the attention of researcher around the globe for many years. Accumulating evidences have indicated that ginsenosides may have great potential in anti-cancer treatments (Qi et al., 2010). However, recent researches and exploration are focused on [Rg.sub.3] and [Rh.sub.2] (Lee et al., 1996; Li et al., 2011; Keum et al., 2003; Kim et al., 2010). Information is very limited about the antitumor activity of [F.sub.2] against gastric cancer cells. The major aim of this study was to investigate the intrinsic molecular mechanism of apoptosis in vitro and in vivo, as well as the probable signaling cascade of chemoprevention induced by [F.sub.2] in SGC7901 cells.
In the mitochondrial pathway, apoptosis results from an intracellular cascade of events in which mitochondrial permeabilization plays a crucial role (Scaffidi et al., 1998). ROS is associated with disruption of MTP, therefore triggering a series of mitochondria-associated events including apoptosis. Mitochondrial dysfunction associated with ROS-mediated cell death induces the pro-apoptotic proteins, such as cyto c or ASK-1, which subsequently causes a caspase-dependent or a JNK-mediated inflammation and apoptosis, respectively (Liu and Tang, 2010). ASK-1 is a redox-sensitive, mitogen-activated protein kinase that is known to induce JNK-mediated inflammation and apoptosis, respectively (Tobiume et al., 2001; Jibiki et al., 2003). Additionally, it has been suggested that JNK has targets in the mitochondria and that mitochondrial JNK activation in response to ROS causes cyto c release (Schwabe and Brenner, 2006). Cyto c activates members of the caspase family, most prominently actives the initiator caspase-9 and the effect or caspase-3 (Thornberry and Lazebnik, 1998). However, ROS induced ASK-1 and its down-stream signaling pathways was rarely reported for the mechanism of ginsensides before. In this study, we demonstrated that the collapse of MTP and accumulation of ROS might activate the ASK-1/JNK pathways, including the elevation of ASK-1, JNK and inhibition of Bcl-2, which trigger the caspase-dependent apoptotic downstream pathway.
Ginsenosides or extract of Panax ginseng as an adjunct to chemotherapy have been evaluated in many types of cancer cells in vitro (Lee et al., 1996; Li et al., 2011; Mao et al., 2012; Keum et al., 2003; Kim et al., 2010; Qi et al., 2010; Yoon et al., 2012). The in vivo data about its promising role as modulator of chemotherapy is limited. It was reported that the survival time of mice transplanted with Hepl-6 cells was significantly longer than that in the control group, after treated i.p. [Rg.sub.3] (3.0 mg/kg) and cyclophosphamide (20.0 mg/kg) for 10 days (Jiang et al., 2011). Other studies have shown that treatment with extract of Panax quinquefoliun at a dosage of 1 g/kg/day demonstrated a marked suppression on tumor volume of Lewis lung carcinoma cells (LLC-l)-bearing mice. C. Hu et al. have proved that IH901, an intestinal metabolite compound K, could inhibited the tumor formation of human gastric carcinoma cell in nude mice at a dose of 10 mg/kg (Hu et al., 2012). The intravenous injection of [F.sub.2] suppressed the growth of tumor formed by intracranial injection of human glioblastoma cells (U373MG) into the right forebrain of SD rat at the dosage of 35 mg/kg. But toxicity of IH901 and [F.sub.2] was not evaluated (Shin et al., 2012). In the present study, we used a smaller dosage of 1.6 mg/kg [F.sub.2] in SGC7901 cells xenograft mouse model. Our results showed significant apoptotic cells in tumor tissue under TUNEL staining. In immunohistochemical staining, 1.6 mg/kg treatment group showed depressed expression of Bcl-2, PARP and increased expression of cyto c caspase 9 and caspase 3. Enhanced side effects are always accompanied by good therapeutic effect in some therapeutic regimens, so the changes in body weight compared with DDP were evaluated during the therapy. No significant drug-induce weight loss was observed in the therapy group, which indicated that [F.sub.2] has low toxicity.
In the present study, our results indicate that Ginsenoside [F.sub.2] might induce apoptosis in tumor cells through the accumulation of ROS and activation of downstream ASK/JNK pathway in SGC7901 cells. Our results provide new information about Ginsenoside [F.sub.2] as a novel chemotherapeutics candidate in gastric cancer.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Received 19 July 2013
Received in revised form 3 September 2013
Accepted 11 October 2013
We thank Dr. Ebony Gary for English editing. This work was supported by the Natural Science Foundation of China (No. 81274018 and 81274068).
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Qian Mao (a,1), Ping-Hu Zhang (b,1), Qiang Wang (c,*), Song-Lin Li (a,*)
(a) Department of Pharmaceutical Analysis & Metabolomics, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, PR China
(b) Jiangsu Center for New Drug Screening Sr National New Drug Screening Laboratory, China Pharmaceutical University, Nanjing, PR China
(c) State Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing, PR China
* Corresponding authors. Tel.: +86 02585639640; fax: +86 85639617.
E-mail addresses: qwang49@l 26.com (Q, Wang), songlinIi64@l 26.com (S.-L Li).
(1) The two authors are co-authors.
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|Author:||Mao, Qian; Zhang, Ping-Hu; Wang, Qiang; Li, Song-Lin|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Mar 15, 2014|
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