Puerarin, isolated from pueraria lobata (Willd.), protects against hepatotoxicity via specific inhibition of the TGF-[beta]1/Smad signaling pathway, thereby leading to anti-fibrotic effect.
Hepatic stellate cell
Recently, the TGF-[beta]1/Smad signaling pathway has been investigated in the pathogenesis of hepatofibrosis, and pharmacological treatment of liver fibrosis targeted this pathway to determine its contribution to the inhibition of fibrotic development. Importantly, ethnopharmacology-derived Pueraria lobata has been reported to effectively reverse the fibrotic process in the liver. In the present study, we performed dimethylnitrosamine (DMN)-induced liver fibrosis in rats to assess the benefits of puerarin (PR), which was isolated from Pueraria lobata (Willd.), on ECM-derived hepatocytes associated with the TGF-[beta]1/Smad pathway. Our results showed that the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN), type III precollagen (PCIII) and type IV collagen (CIV) were significantly reduced by PR treatment, while hepatic homogenates showed decreased levels of hydroxyproline (Hyp) and collagen I (Coil). Masson's trichrome staining indicated that the DMN-induced liver fibrosis was alleviated. In addition, the protein expression levels of transforming growth factor-[beta]I (TGF-[beta]l), smad2, smad3, ot-SMA and TIMP-1 were downregulated specifically by PR treatment, whereas the protein expression levels of smad7 and MMP-1 were upregulated. Furthermore, we evaluated the PR-mediated inhibitory effect on TGF-[beta]1-treated proliferation and activation in a rat liver stellate cell line (HSC-T6). These data resulted in inhibition of the cell growth of HSC-T6 in a dose-dependent manner and a reduction in TRI, smad2 and smad3 expressed proteins in the presence of PR on TGF-[beta]1-treated HSC-T6 cells, while smad7 levels were downregulated. Taken together, these findings identify a unique effect for PR-regulation of the TGF-[beta]1/Smad pathway in blocking fibrotic development and provide a promising strategy for hepatofibrosis treatment.
[c] 2013 Elsevier GmbH. All rights reserved.
Hepatic fibrosis is a pathophysiological progression associated with excessive extracellular matrix (ECM) deposition in response to chronically damaged liver tissue (Bataller and Brenner 2005; Lee and Friedman 2011). The formation of hepatic fibrosis in correlative fibrous connective tissue is a reparative or reactive process (Anty and Lemoine 2011). Hepatic stellate cells (HSCs), the major cell type involved in liver fibrosis, play a pivotal role in ECM remodeling and hepatic fibrosis development. When the liver is lesioned, HSCs are activated and play subsequent roles in proliferation, contractility and chemotaxis. Activated HSCs can also secrete collagen constituted scar tissue, which can result in cirrhosis (Sarem et al. 2006; Li et al. 2013a, b). Although the precise pathogenic mechanisms remain unclear, several medications have been used to treat clinical liver fibrosis. Silymarin therapy with the effective dosage can mitigate the symptoms related to hepatopathy. Silymarin has also been reported to protect against cytotoxicity in hepatocytes (Sailer et al. 2001; Feller and Lengyel 2008). In addition, multiple drug therapies have been used to treat hepatic fibrosis with some success, including toxicity reduction and efficacy reinforcement (Sun and Li 2000; Lian et al. 2005; Zhang 2012). Phytomedicine refers to traditional medicine in which the herbs used are scientifically validated and are categorized as natural medicines. In traditional Chinese medicine (TCM), Pueraria lobata has traditionally been used as a remedy for alcoholism and hangovers in China, where it is thought to detoxify the liver and alleviate its subsequent symptoms (McGregor 2007; Penetar et al. 2011). Puerarin lobata or its extracts have been promoted as a supplement or therapy for a diversity of symptoms, including headaches, alcoholism, intestinal obstruction, diarrhea, angina pectoris and hypertension (Lukas et al. 2005; Wong et al. 2011; Yang et al. 2010). In addition, our previous studies found that puerarin (PR) resulted in attenuation of hepatofibrosis via a reduction in ECM deposition in rats with C[CI.sub.4]-induced hepatic fibrosis (Li et al. 2013 a, b). However, PR-targeted TGF-[beta]1/Smad signaling pathway involved in hepatofibrosis progression has remained largely unexplored.
Liver fibrosis is also tightly associated with the TGF-[beta]1/Smad signaling pathway at multiple levels. Thus, we proposed that PR might effectively exert resistance to collagenation by mediating this signaling pathway. In this study, we examined the expression of collagenic components using in vivo and in vitro studies involved in the PR-mediated regulation of the TGF-[beta]1/Smad signaling pathway, and demonstrated the anti-fibrotic effect of PR and its underlying mechanism.
Materials and methods
PR (purity > 99.0%; the structural formula is shown in Fig. 1) was provided by the Department of Pharmaceutical Chemistry, Guangxi Medical University. Silymarin (purity >95.0%) was purchased from the Xi'an Rongsheng Biotechnology Co., Ltd. (Xi'an, China). Analytical grade dimethylnitrosamine was purchased from the Chengdu Kelong Chemical Reagent Factory (Chengdu, China). Other chemicals and reagents were labeled as described below.
Acute toxicity testing
Ten Sprague Dawley (SD) rats were fasted for 16 h and the animals' body weight was recorded pre-treatment. A specific dose of PR (final concentration: 500 mg/ml; 6000 mg [kg.sup.-1] body weight) was orally administered to each rat and changes in the behaviors, vital signs and mortality were observed.
Animals and drug intervention
Six-week-old healthy male SD rats weighing approximately 200 [+ or -] 20g were purchased from the Medical Laboratory Animal Center of Guangxi Medical University, China (Certificate No. SCXK-Gui-2009-0002). The study was conducted according to protocols approved by the Institutional Ethical Committee of Guilin Medical University. All of the rats were housed under controlled conditions with a temperature of 25 [+ or -]2[degrees]C, relative humidity of 60 [+ or -] 10%, room air changes 12-18 times/h, and a 12-h light/dark cycle. The study was conducted in accordance with the U.S. guidelines (NIH publication #85-23, revised in 1985) for laboratory animal use and care.
To screen the fibrotic process, the rats were randomly divided into two groups: control group and hepatic fibrosis model group. Hepatic fibrosis animals intraperitoneally received 0.6 ml [kg.sup.-1] (for a period of 1 week) and 1.2 ml [kg.sup.-1] (for a period of 5 week) 0.5% DMN (dissolved in distilled water) twice a week for 6 consecutive weeks. The control animals received an equivalent volume of saline twice a week. Two rats from model group and one rat from control group were randomly chosen, and a pathological examination was performed to monitor the formation of hepatofibrosis at week 2, 4 and 6, respectively.
Rats were randomly assigned into six groups: Group I (n =10): normal rats were administered normal saline and used as normal controls; Group II (n=20): fibrotic rats were administered normal saline and used as model controls; Group III (n = 20): fibrotic rats were administered Silymarin (SM, 400 mg [kg.sup.-1] body weight) by oral gavage once a day for 4 weeks; Group IV (n =20): fibrotic rats were administered PR (200 mg [kg.sup.-1] body weight) by oral gavage once a day for 4 weeks; Group V (n =20): fibrotic rats were administered PR (400 mg [kg.sup.-1] body weight) by oral gavage once a day for 4 weeks; Group VI (n=20): fibrotic rats were administered PR (800 mg [kg.sup.-1] body weight) by oral gavage once a day for 4 weeks.
Analysis of functional liver enzymes
The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), hyaluronic acid (HA), laminin (LN), type ill precollagen (PCIII) and type IV collagen (CIV) were measured using commercially available kits (Wuhan Boster Bio-Engineering Limited Company, Wuhan, China) and performed according to the manufacturer's protocol.
After 4 weeks of PR administration, all of the rats were anesthetized with 20% urethane, and the serum samples were collected and stored in tubes containing heparin. Liver specimens were dissected and washed immediately with ice-cold saline to remove excessive blood. In addition, some of the specimens were properly conserved for subsequent tests. Other specimens were fixed in 10% formal in solution for subsequent staining procedures.
Measurement of liver index
Liver index = (Liver weight/Body weight) x 100%,
Thymus index = Thymus weight/Body weight,
Spleen index= Spleen weight/Body weight.
Liver tissue samples (n= 10, each group) were sectioned, and Masson's trichrome staining procedure was performed. The pathological changes were monitored and imaged under a light microscope (Olympus, CX41, Japan). In addition, the morphological changes of the liver lesion were evaluated using the following criteria: score 0, absent fibrosis; score 1, presence of fibrosis; score 2, mild fibrosis; score 3, moderate fibrosis; and score 4, severe fibrosis (Li et al. 2013a,b).
Estimation of liver collagenic parameters
Hepatic tissues were washed with normal saline to remove excess blood and clots. The tissues were then homogenized on ice with Tris-HCI (6 mmo1/1) containing 3 mmo1/1 EDTA (pH 7.2). Homogenates were centrifuged at 1500 x g for 10 min at 4[degrees]C. Hydroxyproline (Hyp) and collagen I (Col I) were determined via radioimmunoassay (RIA) using commercially available kits (Maker Science and Technology Co., Ltd., Sichuan, China) according to the manufacturer's instructions.
Western blotting analysis
Briefly, proteins were extracted from hepatic tissue samples, separated using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and then transferred onto PVDF (polyvinylidene fluoride) membranes under ice-cold conditions. The membranes were then incubated with primary antibodies against TGF-[beta]I, smad2, smad3, smad7,[alpha]-SMA, TIMP-1 and MMP-1 (1:1 000; Santa Cruz, USA). [beta]-Actin antibody was used as a loading control. After washing, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (l-IRP) (1:2000; Santa Cruz, USA). Signals were detected using the enhanced chemiluminescence (ECL) detection system (Pierce, USA), and the protein expression levels were determined using Quantity One software (Bio-Rad, USA).
The HSC-T6 cell line was obtained from American Type Culture Collection. HSC-T6 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS) (1% peni-cillin/streptomycin containing 10.000 U/mIpenicillin G sodium and 10,000 mg/ml streptomycin sulfate). The cells were cultured at 37[degrees]C in a 5% C[O.sub.2] incubator.
Anti-proliferative activity using an M'TT assay
Cell proliferation following PR treatment was measured using an MTT assay. HSC-T6 cells were seeded at a density of 1 x [10.sup.5] cells/100 [micro]l/well in 96-well plates. The medium was then removed and 150 [micro]l DMEM containing FBS was added to the TGF-[beta]l revulsant (final concentration adjusted to 6 ng/ml) with increasing stepwise concentrations of PR (50, 100, 200 nM), TGF-[beta]I specific inhibitor SB-431542 (100 nM) (Sigma, USA) and a blank control. After the co-incubation time, 20[micro]l MIT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma, USA) was added to each well containing the culture medium, and the samples were further incubated for 4h at 37[degrees]C. The medium was then removed and 1O0-[micro]l lysate was added to each well with gentle shaking for 10 min. The optical density for each well was measured at 570 nm using an ultraviolet spectrophotometer (UV-2600, Shimadzu, Japan).
The results were calculated using the following formula:
% inhibition((OD control--OD blank)--(OD treated--OD blank))/(OD control--OD blank)x100
Western blotting analysis
Total proteins were extracted with RIPA lysis buffer (Sigma, USA) from cultured HSC-T6 cells at 72 h following PR treatment. After fractionation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins (60 [micro]g) were transferred on to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% fat-free milk for 24 h, and then incubated with the following rabbit-monoclonal primary antibodies: TPRI, smad2, smad3 and smad7 (1:1000; Santa Cruz Biotechnology, USA) at 4[degrees]C overnight. After washing three times with Tris-buffered saline Tween-20 (TBST) buffer, the membranes were incubated with a goat-anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Zhongshan Gloclenbridge Biotechnology Co, Beijing, China) at 4 C for 1 h. The intensity of the specific immunoreactive bands was detected using chemiluminescence (ECL) Western blotting detection reagents (Beyotime, Jiangsu, China), and the protein levels were quantified and expressed against control protein.
Statistical analyses were performed using SPSS 13.0 software (SPSS Inc., USA). Comparisons between different groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Data were expressed as the mean [+ or -] SD. p < 0.05 was considered as statistically significant.
Assessment of PR-mediated acute toxicity testing in rats
We first evaluated the maximum dose of PR (6000 mg [kg.sup.-1] body weight) applied to SD rats to monitor for any changes within 48 h. In addition, the median lethal dose (LD50) of PR was greater than 6000 mg [kg.sup.-1] body weight in rats. Thus, the PR administration was considered to be safe.
PR treatment enhanced the survival rate of DMN-treated rats
The number of rats in the DMN-treated group was markedly reduced at the end of 4 weeks post-treatment (Fig. 2), which resulted in a survival rate that was less than the animals in the normal control group (p <0.01, n = 10). Importantly, PR-administered rats showed an increased survival rate in a dose-dependent manner (p<0.01. n=10).
PR treatment reduced the activity of functional liver enzymes
Biochemical data showed that DMN-treated rats induced a pronounced increase in the serum levels of ALT and AST, which was much higher compared to normal rats (p <0.01, n =10). However, the elevated levels of these enzymes were effectively attenuated by the SM and PR treatments (p <0.01, n = 10) (Fig. 3).
PR treatment reversed the effect of increased collagenic accumulation
Our results indicated that DMN administration (liver fibrosis model) increased the serum levels of HA, LN, PCIII and CIV in the liver fibrosis rat (p <0.01, n = 10). Treatment with SM and PR resulted in a gradual decrease in HA, LN, PCIII and CIV activities in liver fibrosis tissue (p < 0.01, n=10). These results suggested that PR treatment inhibited DMN-induced collagenic changes (Fig. 4).
PR treatment decreased visceral indices
The results from the liver index suggested that fibrotic rats showed a pronounced hepatic hypertrophy caused by DMN-induced toxicity and was accompanied by elevated thymus/spleen indices compared to normal rats (p < 0.01, n= 10). Following treatment with SM and PR, the changes present in DMN-treated rats were effectively ameliorated. indicating a decreased liver index and thymus/spleen indices (p <0.01, n = 10) (Fig. 5).
Masson's trichrome staining showed that there were no collagen fibers in the interlobular and portal areas, and few collagen fibers in the central veins and sinusoidal. We found no fibrosis in the normal control group, and the structure of the hepatic lobules was normal (Fig. 6- 1). In contrast, a large number of red and green collagen fibers was observed in the model groups. Hepatic lobules re-segmented the formation of a large number of false lobules in tissue sections. Moreover, collagen fibers interconnected in a broadband-like manner, and cell degeneration or necrosis was significantly observed in the model control group (Fig. 6-II). In both the SM and PR low- and -medium dose treated groups, the extent and area of the collagen fibers, as well as the infiltration of inflammatory cells was reduced (Fig. 6-III-V). In addition, the hepatic parenchyma was well restored with a radial arrangement around the central vein, and the number of hepatocytes was increased in the PR high dose group (Fig. 6-VI).
PR treatment reduced collagen deposition
To investigate the collagenic biomarkers involved in fibrosis, we analyzed the contents of Hyp and Col I in liver tissue. We found that DMN-treatal rats showed significantly enhanced Hyp and Col I expressions, and these changes were notably inhibited by the SM and PR treatments (p <0.01. n =10) (Fig. 7).
PR treatment regulates proteins associated with the TGF-[beta]1/Smad pathway
Next, we investigated the effect of PR on protein expression using Western blotting analyses. Our results showed that in the control group, endogenous TGF-[beta]1, Smads, [alpha]-SMA, TIMP-1 and MMP-1 proteins were expressed in the liver in physiological levels. However, DMN administration significantly increased TGF-131, smad2, smad3, [alpha]-SMA and TIMP-1 expressions, while smad7 and MMP-1 levels were reduced (p <0.01, n =10). Interestingly, the SM and PR treatments nearly reversed these effects on protein expression (p < 0.01, n = 10) (Fig. 8).
PR treatment inhibited the proliferation of HSC-T6 cells
We also evaluated the inhibitory effect of PR on HSC-T6 cell proliferation using the MTT assay. Cell proliferation was inhibited in a dose-dependent and time-dependent manner following increased stepwise concentrations of PR administration. In addition, PR treatments (50, 100,200 nM) significantly attenuated number of HSC-T6 cells (p <0.01) compared to vehicle control (Fig. 9). Specifically, the PR-mediated inhibitory effect on TGF-[beta]1-treated HSC-T6 cells was similar to that of the SB-431542 specific inhibitor, which targets TGF-[beta].
PR treatment downregulated T[beta]RI, smad2 and smad3 expression and upregulated smad7 expression at the protein level
The protein levels of T[beta]RI, smad2 and smad3 in PR-treated HSC-T6 cells was effectively decreased in a time- and dose-dependent manner at 72 h (p < 0.01) (Fig. 10), and the expression of smad7 was specifically elevated at post-treatment (p <0.01). Furthermore, high dose treatment of PR (200 nM) significantly inhibited the changes in protein levels, similar to the effect observed with SB-431542 treatment. In addition, the effect of PR treatment on the regulation of these proteins was consistent with the anti-proliferative effects observed in the MIT assay.
Hepatic fibrosis is a common pathological basis of hepatic cirrhosis and liver cancer and is characterized by liver function failure (Jiao et al. 2009). The extracellular matrix (ECM) is mainly composed of collagen proteins. ECM components (such as HA, LN, PCIII and CIV) may be altered by metabolic collagen, which corresponds with the degree of hepatic fibrosis (Ruehl et al. 2011). Elevated serum levels of ALT and AST induced by chemical hepatotoxicity are associated with liver structural lesions, which results in functional liver enzymes that are abnormally present in the cytoplasm and released into circulation (Chen et al. 2000: Meng et al. 2010). Thus, the level of liver enzymes is important for diagnosis by clinicians and reflects liver function (Giannini et al. 2005). In the present study, DMN-induced liver fibrosis resulted in increased serum levels of ALT and AST and was accompanied by increased visceral indices (including the liver, thymus, and spleen tissues), indicating liver dysfunction and an immunocompromised status. After treatment with PR, the serum enzymatic levels and visceral indices were significantly lowered, suggesting that PR therapy improved liver function and immune competence in DMN-lesioned rats. In addition, experimental application of Masson's trichrome enabled the observation and analysis of liver fibrosis development. These results showed that PR treatment could effectively reverse liver fibrosis processes in the presence of DMN.
As one of the leading causes of chemically induced liver injury or fibrosis, collagen is gradually deposited via continual circulation (Weber et al. 2003). Counteractivity or inhibition of collagenation may help antagonize against liver fibrosis development (An et al. 2012). Several alternative medicines, which attenuate other types of ECM components, have shown therapeutic benefits against liver damage (Kinnula 2005). In this study, PR treatment rescued the increased serum levels of collagenic components (HA, LN, PCIII and CIV), and Hyp and Col I levels in the DMN-lesioned liver. These data indicated that PR might help prevent against unregulated collagenic generation and accumulation. In addition, the benefit of PR treatment contributed to the amelioration of collagenic metabolism in liver tissue. Thus, we investigated the PR-mediated anti-fibrotic effects associated with a signaling pathway in hepatocytes.
Several reports have suggested that TGF-[beta], a powerful signaling mediator, primarily activates hepatic stellate cells (HSCs), Kupffer and perisinusoidal cells, which are cells that have been implicated in hepatic fibrosis processes (Tahashi et al. 2002). Moreover, TGF-[beta]1 has been publicly recognized as one of most important promoting factors for HSC activation (Williams and Ireclale 2000). In addition, TGF-[beta]1 plays a significant role in HSCs activation via stimulation of the synthesis and secretion of Type-1 or Type-2 collagen and fibronectin to promote extracellular matrix (ECM) formation (Zhou et al. 2010; Baarsma et al. 2011). In addition, TGF-[beta]1 demonstrates important effects such as inhibition of MMP activity, enhancement of TIMP activity, such as matrix metalloproteinase (MMP) inhibitors, enhancement of the expression of ECM receptors, and promotion of ECM adhesion in cells (Liu et al. 2006; Gomes et al. 2012). However, Smads are intracellular proteins that transmit extracellular signaling from TGF-[beta] ligands into the nucleus where they may activate downstream TGF-[beta] gene transcription (Miyazono et al. 2000). Numerous overexpression studies have shown that the levels of smad2 and smad3 proteins dramatically increase in an animal model of liver fibrosis, while smad7 inhibits experimental fibrogenesis in vivo via activation of an anti-liver fibrosis signal (Flanders 2004; Uemura et al. 2005; Dooley et al. 2003). Consequently, liver fibrosis processes may be significantly mitigated when the synthesis of TGF-[beta]1 and/or the TGF-[beta]1 /Smacl signaling pathway are blocked. Our results indicated that DMN-lesioned rats resulted in an increased expression of TGF-[beta]l, Smads, [alpha]-SMA, TIMP-1 and MMP-1 proteins in hepatocytes, suggesting that DMN-induced hepatotoxicity was associated with the activation of the TGF-[beta]1/Smal pathway and its downstream effectors. Interestingly, we observed that endogenous hepatic TGF-[beta]1 and Smads levels were effectively attenuated by PR treatment in a dose-dependent manner, and subsequently reversed lesion formation in liver tissue. Our findings indicated that PR-mediated cytoprotection was associated with TGF-[beta]l /Smad pathway inactivation, which was accompanied by concomitant decreases observed in the hepatocytes of DMN-lesioned rats. Thus, we extrapolated that PR inhibited the proliferation and activation of ECM-derived hepatocytes (such as HSCs, fibroblasts, Kupffer cells) via inactivation of the endogenous TGF-[beta]1/Smad pathway in fibrotic rats.
Next, we further determined whether PR-inhibited ECM-derived hepatocytes in cultured HSCs were involved in the signaling pathway. Consistent with findings in vivo, our results showed that the PR-mediated inhibitory effect on TGF-[beta]1-treated HSC-T6 cells resulted in a reduction of VRI, smad2 and smad3 levels and an elevation of smad7 expression. Interestingly, these results also indicated that PR suppressed the overproliferation of HSCs via TGF-[beta]1 pre-treatment, most likely via the blockade of TGF-[beta]1/Smad-dependent signaling and thereby attenuating collagenic generation and deposition.
Taken together, our results demonstrate that PR therapy is a potential therapeutic candidate for the management of hepatofi-brosis due to its fibrinogenolytic effect on fibrotic development. Importantly, PR-mediated hepatoprotection is associated with specific inhibition of the TGF-[beta]/Smad signaling pathway in HSCs.
Conflict of interest
The authors declare no conflicts of interest and are responsible for the contents of this report.
We are grateful to Prof. Jiaquan Li and Prof. Haiyuan Xie for their excellent technical supports.
* Corresponding author at: Faculty of Basic Medicine. Guilin Medical University, 109 Huanchengbei Road Two, Guilin, Guangxi 541004, PR China.
Tel.: +86 773 5895291; fax: +86 773 5895278.
** Corresponding author at: College of Stomatology of Guangxi Medical University, 10 Shuangyong Road, Nanning, Guangxi 530021, PR China.
Tel.: +86 771 5336742; fax: +86 771 5357938.
E-mail addresses: firstname.lastname@example.org (R. Li), liangtao_gxmuail 26.com (T. Liang).
(1) These authors contributed equally to this work.
0944-7113/$--see front matter [c] 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.06.008
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Lingyuan Xu (a), (1), Ni Zhengc (b), (1), Qiaoling He (d), Rong Li (b) *, Kefeng Zhang (b), Tao Liang (c), **
(a) Department of Pharmacy, The Affiliated Hospital of YouJiang Medical University for Nationalities. Baise 533000. PR China
(b) Guilin Medical University, Guilin, Guangxi 541004, PR China
(c) Drugs and Devices Department, College of Stomatology of Guangxi Medical University, Nanning 530021, PR China
(d) Guangxi Medical University, Nanning 530021, PR China
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|Author:||Xu, Lingyuan; Zheng, Ni; He, Qiaoling; Li, Rong; Zhang, Kefeng; Liang, Tao|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Oct 15, 2013|
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