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Protective effect of Wuzhi tablet (Schisandra sphenanthera extract) against cisplatin-induced nephrotoxicity via Nrf2-mediated defense response.


Cisplatin is a potent anti-cancer agent for various types of tumors. However, the clinical use of cisplatin is often limited by its nephrotoxicity. This study reports that WZ tablet (WZ, a preparation of an ethanol extract of Schisandra sphenanthera) mitigates cisplatin-induced toxicity in renal epithelial HK-2 cells and in mice. Pretreatment of HK-2 cells with WZ ameliorated cisplatin-induced cytotoxicity caused by oxidative stress, as was demonstrated by reductions in the levels of reactive oxygen species (ROS) and increased levels of glutathione (GSH). WZ facilitated the nuclear accumulation of the transcription factor NF-E2-related factor 2 (Nrf2) and the subsequent expression of its target genes such as NAD(P)H:quinine oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1) and glutamate cysteine ligase (GCL). Protective effects of WZ on cisplatin-induced nephrotoxicity were also observed in mice. WZ attenuated cisplatin-induced renal dysfunction, structural damage and oxidative stress. The nuclear accumulation of Nrf2 and its target genes were increased by WZ treatment. Taken together, these findings demonstrated WZ have a protective effect against cisplatin-induced nephrotoxicity by activation of the Nrf2 mediated defense response, which is of significant importance for therapeutic intervention in cisplatin induced renal injury.


Wuzhi tablet

Schisandra sphenanthera





Cisplatin is one of the most effective chemotherapeutic agents for the treatment of various cancers including those of the breast, ovarian, lung, bladder, esophageal, and head, etc. However, the clinical utility of cisplatin is limited by its nephrotoxicity with about 2530% of patients experiencing a significant decline in renal function after a single dose of cisplatin (Pabla and Dong 2008). Accordingly, the prevention of nephrotoxicity is an important aspect of cisplatin chemotherapy. The mechanism of cisplatin induced nephrotoxicity is not completely clear. In vitro and in vivo evidence have suggested that oxidative stress plays a critical role in the pathogenesis of cisplatin induced nephrotoxicity (Perez-Rojas et al. 2009). Oxidative stress is caused by various free-oxygen radicals including superoxide anion, hydrogen peroxide and hydroxyl radical. Excessive production of free radicals and the occurrence of lipid peroxidation due to oxidative stress are implicated in cisplatin-induced renal dysfunction (Chirino et al. 2008).

A number of signaling pathways, most remarkably those controlled by the transcription factor Nrf2, are activated to counteract accumulating reactive oxygen species and electrophiles (Aleksunes et al. 2010; Zuniga-Toala et al. 2013). Under normal conditions, Nrf2 is present in the cytoplasm in association with the Kelch-like ECH-associated protein 1 (Keap1), which functions as a negative regulator of Nrf2 by retaining it in the cytosol and enhancing its proteasomal degradation. Exposure to pharmacological activators or generation of oxidative stress, Nrf2 translocate to the nucleus where it transactivates a battery of genes encoding various antioxidant phase II detoxifying enzymes by binding to antioxidant response elements (ARE) (Li et al. 2012b). The antioxidant phase II detoxifying enzyme plays a major role in the detoxification of ROS produced by xenobiotics. Recent evidence has suggested that activating Nrf2-dependent cellular defense system may provide a therapeutic target against cisplatin-induced oxidative injury (Aleksunes et al. 2010; Moon et al. 2013).

Wuzhi tablet (WZ) is a preparation of an ethanol herb extract of Wuweizi (Schisandra sphenanthera), which contains 7.5 mg Schisantherin A per tablet. Its major active chemical constituents include Schisandrin A, Schisandrin B, Schisandrin C, Schisandrol A, Schisandrol B, Schisantherin A, and Schisantherin B. WZ is a prescribed drug (Registration number in China: Z20025766) in clinical practice for the treatment and prevention of many diseases such as hepatitis, inflammation and cancer (Huyke et al. 2007; Jin et al. 2011). To guarantee the quality of the WZ tablet, our research group has further determined the content of the major lignans in WZ including Schisantherin A, Schisandrin A, Schisandrin B, Schisandrin C, Schisandrol A, and Schisandrol B using a validated LC-MS/MS analysis as described in the previously published paper (Qin et al. 2013). Previous studies reported that WZ or the compounds extracted from WZ protected against acute liver injury due to free radical scavenging effect, inhibition of lipid peroxidation, and increased antioxidant activity (Cheng et al. 2013). To date, whether WZ could protect against cisplatin induced nephrotoxicity has not been investigated yet. This study was designed to investigate the protective effect of WZ on cisplatin induced renal injury and to examine whether Nrf2/ARE pathway was activated by WZ treatment. This study is of significant importance for therapeutic intervention in cisplatin induced nephrotoxicity.

Material and methods


Wuzhi tablets (batch number 110711, drug approval number in China: Z20025766) were manufactured and provided by Fanglue Pharmaceutical Company (Guangxi, China) under GMP guidelines. The 70% ethanol extract of S. sphenanthera fruit was formulated with starch, carboxymethyl starch sodium, and magnesium stearate into tablets. The final product meets the China SFDA standard (YBZ14932006) and has been quantified to 7.5 mg Schisandra lignans per tablet by UV analysis. The fingerprint analysis of Wuzhi tablet was reported in our recently published paper (Qin et al. 2014). In addition, contents of major active constituents in the extract including Schisantherin A, Schisandrin A, Schisandrin B, Schisandrin C, Schisandrol A and Schisandrol B were further identified and determined in our laboratory using a validated LC-MS/MS analysis (Qin et al. 2013). As described in our previously published paper, the contents of Schisantherin A, Schisandrin A, Schisandrin B, Schisandrin C, Schisandrol A and Schisandrol B in Wuzhi tablet extracted by ethanol were 10.089, 7.244, 0.017, 0.024, 0.048 and 0.468 mg/g, respectively (Qin et al. 2013).

Cisplatin, sulforaphane (SFN) and anti-[alpha]-tubulin antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-Nrf2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Histone H3 antibody was obtained from Cell Signaling Technology (Beverly, MA, USA). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), trypsin, antibiotic (10,000 U/ml penicillin and 10,000 [micro]g/ml streptomycin) and other tissue culture reagents were purchased from Gibco. All other chemicals were analytical grade and commercially available.

Preparation of samples

The WZ extract used in the in vitro experiment was prepared according to our previously published study (Qin et al. 2010). Briefly, 30 times (V: W) of ethanol (ml) was added into pulverized powder of WZ tablet (g) and vortexed for 1 min. The mixtures were then ultrasonic-extracted for 1 h and then centrifuged for 10 min. The residue was dissolved in ethanol. The above steps were repeated again. The supernatants were combined and transferred to a clean tube and evaporated to dryness under vacuum. The residue was dissolved in ethanol as the stock solution (0.4 g/ml), and stored at -20[degrees]C until use. In the in vivo study, the pulverized powder of WZ tablet was suspended in the distilled water and intragastrically given to mice.

Cell culture and treatments

HK-2 cells, an immortalized human renal proximal tubule epithelial cell line, were kindly provided by Prof. Xueqing Yu (Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, PR China). The cells were cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F12, Gibco Laboratories, Grand Island, New York, USA) supplemented with 10% fetal bovine serum (FBS, HyClone) and antibiotics (50 U/ml of penicillin and 50 [micro]g/ml streptomycin). The cells were grown in a humidified incubator with 5% C[O.sub.2] at 37[degrees]C.

Animals and treatments

Male NIH mice (weighing about 18-20 g) were obtained from the Laboratory Animal Center of Sun Yat-sen University. The animals were kept in a room at 22-24[degrees]C with a light/dark cycle of 12/12 h and 55-60% relative humidity. They had free access to standard rodent chow and clean tap water. All procedures were approved by the Animal Ethics Committee of Sun Yat-sen University, in accordance with National Institute of Health and Nutrition Guidelines for the Care and Use of Laboratory Animals.

Animals were divided into four groups of ten animals each and treated as the following: (1) control group: gavaged with saline (5 ml/kg) for 10 consecutive days and a single intraperitoneal injection of saline (5 ml/kg) on the 7th day; (2) WZ group: gavaged with WZ (0.5 g/kg) for 10 consecutive days and a single intraperitoneal injection of saline (5 ml/kg) on the 7th day; (3) cisplatin group: gavaged with saline (2 ml/kg) for 10 consecutive days and a single intraperitoneal injection of cisplatin (15 mg/kg) on the 7th day; (4) WZ + cisplatin group: gavaged with WZ (0.5 g/kg) for 10 consecutive days and a single intraperitoneal injection of cisplatin (15 mg/kg) on the 7th day. The dosages for WZ and cisplatin were selected based on previous studies (Bi et al. 2013; Guerrero-Beltran et al. 2010). Animals were sacrificed at day 10, which was 72 h after cisplatin administration. Blood samples were collected to evaluate serum creatinine and blood urea nitrogen (BUN) levels. One kidney was fixed in 10% formalin for histopathological studies, and the other kidney was immediately frozen in liquid nitrogen and stored for future molecular biological analysis.

Quantitative RT-PCR

Total RNA was prepared using the RNAiso Plus (TaKaRa, Japan) according to the manufacturer's protocol. The quantity and purity of RNA were assessed by absorbance at 260 and 280 nm. The cDNA was prepared from the total RNA (1 [micro]g) with a reverse transcriptase (RT) Primer Mix using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer's instructions. The primers for real-time PCR analysis were shown in Table 1. The subsequent PCR amplification was carried out on a LightCyder 2.0 system (Roche Diagnostics, Basel, Switzerland) using 40 or 45 cycles of 95[degrees]C for 5 s and 60[degrees]C for 20 s. G was used as an internal control. Fold changes, expressed as the mean [+ or -] standard deviation (SD), were calculated for the treated groups vs. the vehicle control using the [2.sup.-[DELTA][DELTA]CT] method.

Western blotting

Nuclear extracts were prepared using a Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's recommendations. Equivalent amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore Co., Billerica, MA, USA). After being blocked in 5% non-fat milk in TBST [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20] for 2 h at room temperature, the membranes were incubated with the primary antibodies rabbit anti-Nrf2 (1:500) and rabbit anti-histone H3 (1:2000) at 4[degrees]C overnight(Li et al. 2012a). The immunoblots were then incubated with a secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The membranes were developed using an electrochemiluminescence (ECL) kit (Thermo Scientific/Pierce, Rockford, IL, USA) according to the manufacturer's protocol. The signals were detected by a chemiluminescence detection system (Bio-Rad Laboratories, Hercules, CA, USA). The density of the immunoreactive bands was analyzed using ImageJ 1.41 (National Institutes of Health, Bethesda, Maryland, USA).

Cell viability assay (MTT assay)

Cell viability was determined by a quantitative colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-um bromide (MTT) (Hsin et al. 2006). HK-2 cells were seeded onto 96-well plates at a density of 5000 cells/well and cultured at 37[degrees]C for 24 h. Cells were pretreated with various concentrations of WZ (2.0, 5.0,10 and 20 [micro]g/ml) for 12 h, and then exposed to cisplatin (9 [micro]g/ml) for 24 h. After treatment, 20 [micro]l of MTT solution (5 mg/ml) was added to the wells and incubated at 37[degrees]C for 4 h. After the incubation, 150 [micro]l of DMSO was added to each well, and the plates were agitated for 10 min. Finally, the absorbance of the resultant solution was measured at 490 nm using a microplate reader (Thermo Multskan Ascent 354, USA).

Determination of cell integrity (LDH leakage assay)

Lactate dehydrogenase (LDH) is an enzyme widely present in the cytosol that converts lactate to pyruvate. When plasma membrane integrity is disrupted, LDH leaks into the culture media, and its extracellular level is elevated (Korzeniewski and Callewaert 1983). HK-2 cells were pretreated with different concentrations of WZ (2.0, 5.0, 10 and 20 [micro]g/ml) for 12 h and then exposed to cisplatin (30 [micro]M) for 24 h. The LDH activity was determined by a LDH Detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions. Enzyme activity was expressed as the percentage of extracellular LDH activity relative to the total LDH activity.

Determination of total CSH

For the measurement of intracellular GSH content, HK-2 cells (3.0 x [10.sup.5] cells/well) were seeded in 6-well plates and grown overnight. After treatment, the cell pellets were lysed by ultrasonication. Following centrifugation (4 x [10.sup.3] g for 10 min at 4[degrees]C), the supernatant (cell extract) was maintained on ice until assayed for the cellular GSH by a GSH detection kit (Nanjing Jiancheng Bioengineering institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions. Total GSH content in mouse kidney tissue was also measured using GSH detection kits (JianCheng Bioengineering Institute, Nanjing, China).

Determination of ROS production

To determine the intracellular accumulation of ROS, the fluorescent probe DCFH-DA was utilized as previously described (Yu et al. 2011). HK-2 cells were pretreated with WZ (20 [micro]g/ml) for 12 h and then exposed to cisplatin (9 [micro]g/ml) for 2 h. After treatment, cells were rinsed with PBS and exposed to 10 [micro]M of DCFH-DA. After incubation for 30 min at 37[degrees]C, the ROS levels were detected using a Monochromators Based Multimode Microplate Reader (Infinite Ml 000, TECAN) at an excitation wavelength of 488 nm and emission wavelength of 525 nm. The raw data from each individual experiment were normalized to vehicle-treated cells.

Measurement of renal lipid peroxidation

Malondialdehyde (MDA), an indicator of ROS production, is one of the end products of lipid peroxidation. MDA was measured in tissues as described previously (Pan et al. 2009). Briefly, renal tissues were homogenized in 0.1 M sodium phosphate buffer (pH 7.4). A reaction mixture consisting of 1.5 ml of 0.8% thiobarbituric acid, 200 [micro]l of 8.1% SDS, 1.5 ml of 20% acetic acid, pH 3.5, and 600 [micro]l of distilled [H.sub.2]O was added to 0.1 ml of processed tissue sample, and the mixture was then heated at 95[degrees]C for 60 min. After being cooled with tap water, the samples were centrifuged at 10,000 g for 10 min at 4[degrees]C and the absorbance of the supernatant was measured at 532 nm with 1, 1, 3, 3-tetramethoxypropane as an external standard. The protein content was determined using a Bradford Protein Assay kit (Beyotime, Jiangsu, China), using BSA as a standard. The level of lipid peroxides was expressed as nanomoles of MDA per milligram of protein.

Histology and histomorphometry evaluation

Kidney specimens were sectioned in blocks and fixed in 10% formalin. After fixation, tissues were dehydrated with a graded series of ethanol and xylene, embedded in paraffin, cut into 3 [micro]m sections, and stained with Mayer' hematoxylin and eosin (H&E). Tubular damage in H&E-stained sections was examined under a light microscope (DM5000B, Leica, Germany) (200 x magnification).

Statistical analysis

SPSS v16.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The values were expressed as the mean [+ or -] SD. Statistical comparisons were made using Student's t-test and one-way analysis of variance (ANOVA), followed by Tukey's test. The level of significance was set at P < 0.05.


WZ protects against cisplatin-induced cell damage

To investigate the effect of WZ on cisplatin-induced damage in HK-2 cells, cell viability and plasma membrane damage were estimated by measuring MTT production (Fig. 1A), LDH release into culture medium (Fig. 1B) and morphological changes (Fig. 1C). Cisplatin (9 [micro]g/ml) significantly reduced cell viability and increased LDH leakage in comparison to control cells. Treatment with WZ (2.0-20 [micro]g/ml) for 24 h resulted in a dose-dependent protection against cisplatin induced cell damage.

WZ decreases ROS production in cisplatin treated HK-2 cells

The generation of ROS plays a pivotal role in the pathogenesis of cisplatin-induced nephrotoxicity. Therefore, the effect of WZ on ROS generation induced by cisplatin was investigated. ROS generation in HK-2 cells was significantly increased after cisplatin treatment compared with the levels of the control (Fig. 2). The increased ROS production was significantly decreased by treatment of WZ.

WZ increases GSH and SOD levels in cisplatin-treated HK-2 cells

GSH is one of the most important endogenous antioxidants protecting cells against oxygen radical damages. SOD catalyzes the dismutation of superoxide radicals and is said to be the primary enzyme that responds oxygen radicals. To investigate the protective effect of WZ against cisplatin induced oxidative injury, the levels of GSH (Fig. 3A) and SOD (Fig. 3B) of treated HK-2 cells were measured. Exposure to cisplatin resulted in a decrease of GSH content and SOD activity, compared with the control. GSH and SOD levels were greater in the groups treated with WZ and cisplatin together than in those treated with cisplatin alone.

Effect of WZ on cisplatin-induced renal dysfunction in mice

To investigate the effect of WZ on cisplatin induced renal dysfunction, levels of blood urea nitrogen (Fig. 4A) and creatinine (Fig. 4B) were measured at 72 h after cisplatin administration. Cisplatin administration resulted in severe renal injury, which was attenuated by treatment with WZ. WZ alone had no effects on the BUN and creatinine levels compared with the vehicle-treated group.

Effects of WZ on histopathological changes

Kidney sections from vehicle and WZ treated mice (Fig. 5A and C) had a normal histopathological appearance, whereas those from cisplatin-treated mice (Fig. 5B) showed severe tubular vacuolations, hyaline cast formation and intraluminal necrotic cellular debris. However, treatment with WZ markedly improved the structural changes induced by cisplatin treatment (Fig. 5D).

Effect of WZ on GSH and MDA levels in cisplatin-treated mice

The renal GSH levels declined in cisplatin-treated mice, and the decrease was prevented by WZ treatment (Fig. 6A). In addition, cisplatin induced an increase in MDA levels, which are an indicator of ROS production, and the increase was attenuated by WZ treatment (Fig. 6B).

WZ induces Nrf2 activation in HK-2 cells and mice

The NrG pathway is regarded as one of the most important mechanisms for protection against oxidative stress. Therefore, we investigate whether WZ activates the NrG pathway and induces the expression of NrG-target genes in HK-2 cells and the kidney of mice. The effects of WZ on the NrG nuclear accumulation were detected by western blot analysis. HK-2 cells were treated with WZ (20 [micro]g/ml) for different time periods. The nuclear protein level of NrG was increased after just 2 h treatment and was able to remain elevated up to 24 h (Fig. 7A). Besides, the nuclear accumulation of NrG in the kidney of mice was also increased by treatment with WZ in cisplatin-treated rats, as compared with cisplatin treatment alone (Fig. 8A).

The mRNA expression levels of NrG-target genes including NQO1, HO-1 and GCLC were measured by real time RT-PCR. The mRNA expression of these genes in HK-2 cells after WZ (20 [micro]g/ml) treatment for different times (2, 4, 6, 12 and 24 h) was shown in Fig. 7B. The expression levels of NQO1 and HO-1 were markedly increased after WZ treatment for 2 h and peaked at 2 h. Interestingly, the expression of GCLC started to increase at 2 h after WZ treatment, fell down at 6 h, and then began to increase up to 24 h (6.0-fold induction). Moreover, treatment with WZ for 2 h induced the expression of NQO1, HO-1 and GCLC in a dose-dependent manner (Fig. 7B). The expression of NQO1, HO-1 and GCLC in the kidney of mice was also increased after oral administration of WZ (Fig. 8B).


Several herbs and herbal products have been proven to possess kidney protective functions against cisplatin induced nephrotoxicity. However, there has been no study to evaluate the effect of WZ on cisplatin induced kidney injury. Our study showed that WZ greatly ameliorated cisplatin-induced nephrotoxicity in renal epithelial HK-2 cells and in mice.

Nephrotoxicity is the major limitation in cisplatin based chemotherapy. Our study revealed that treatment with cisplatin resulted in marked nephrotoxicity in vitro and in vivo, which was consistent with previous reports (Ahmad and Sultana 2012; Yu et al. 2009). Oxidative stress is considered to play an important role in cisplatin-induced nephrotoxicity. In agreement with previous studies, treatment of HK-2 cells with cisplatin resulted in ROS generation and administration of WZ decreased the production of ROS. MDA is considered to be one of the end products of lipid peroxidation and an indicator of ROS production. Our results showed that cisplatin treatment induced renal lipid peroxidation, which was paralleled by the deterioration of renal structure and function. Treatment of WZ reduced the production of MDA induced by cisplatin. The generation of oxygen radicals depletes the supply of GSH, leaving tissues vulnerable to the damage by oxygen radicals. Cisplatin has been found to deplete GSH. In this study, WZ restored GSH concentrations in cisplatin treated HK-2 cells and mice. These data suggested that WZ might recover the antioxidant system in the kidney.

The Nrf2 pathway is regarded as one of the most important mechanisms in the cell for protection against oxidative stress generated from exposure to exogenous or endogenous chemicals. Using an unbiased transcriptomic approach Wilmes et al. identified the Nrf2 pathway as the most significant signaling response in renal epithelial cells challenged with nephrotoxin (Wilmes et al. 2011). Aleksunes et al. reported that the absence of Nrf2 exacerbates cisplatin-induced nephrotoxicity in mice and pharmacological Nrf2 activation may be a novel therapeutic strategy to suppress renal injury (Aleksunes et al. 2010). The results showed that WZ activated Nrf2 with the consequent induction of downstream cytoprotective genes including NQO1, HO-1 and GCLC in HK-2 cells and in mice. Activation of NrO is regulated at multiple levels, such as transcriptional control, translational control, Keap1 regulation and phosphorylation. Further study is needed to investigate whether other mechanism are underlying the activation of Nrf2 by WZ.

Several studies have reported that WZ or some components in WZ exhibited antitumor activities (Gao et al. 2013; Huang et al. 2008; Min et al. 2008), which suggested that WZ might exhibit synergistic anticancer effect with cisplatin. Further study is required to investigate the effect of combination use of WZ and cisplatin in tumor xenograft animal models.

In conclusion, our results showed that WZ attenuated cisplatin-induced nephrotoxicity, and this protective effect was associated with the activation of Nrf2/ARE pathway in HK-2 cells and the kidney of mice. Our study provides valuable data that WZ can have significant potential for therapeutic intervention in cisplatin induced nephrotoxicity.

Conflict of interest

The authors declare that there are no conflicts of interest.


This work was supported by National Science Foundation of China (No. 81102886) and Administration of Traditional Chinese Medicine of Guangdong Province of China (No. 20141051).


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Jing Jin (a), Mei Li (a), Zhongxiang Zhao (b), Xiaozhe Sun (a), Jia Li (c), Wenwen Wang (a), Min Huang (a), Zhiying Huang (a), *

(a) School of Pharmaceutical Sciences, Sun Yat-sen University, 132 East Circle at University City, Guangzhou 5 WOOS, PR China

(b) School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China

(c) Pharmaceutical Department, Cancer Center of Guangzhou Medical University, Guangzhou 510095, PR China


Article history:

Received 19 January 2014

Revised 31 December 2014

Accepted 12 March 2015

* Corresponding author. Tel.: +86 20 39943025; fax: +86 20 39943000.

E-mail address: (Z. Huang).

Table 1
Primer sequences for real-time PCR analysis.

Gene    Primer sequences (human)


Gene    Primer sequences (mouse)

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Author:Jin, Jing; Li, Mei; Zhao, Zhongxiang; Sun, Xiaozhe; Li, Jia; Wang, Wenwen; Huang, Min; Huang, Zhiyin
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:May 15, 2015
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