Effects of alisol B 23-acetate on ovarian cancer cells: G1 phase cell cycle arrest, apoptosis, migration and invasion inhibition.
Background: Ovarian cancer is the first leading cause of death among gynecologic malignancies worldwide. Discovery of new chemotherapeutic drugs is still imperative for the improvement of the survival rate.
Purpose: This study aims to investigate the anti-cancer potential of alisol B 23-acetate (AB23), a protostane-type triterpene isolated from the Alismatis Rhizoma, in the parental and paditaxel-resistant ovarian cancer cells.
Methods: MTT assay was performed to evaluate cell viability after treatment with AB23, along with flow cytometry for apoptosis and cell cycle analysis. Western blotting was conducted to determine the relative protein level. Wound healing and transwell assays were performed to investigate the effect of AB23 on cell migration and invasion.
Results: AB23 obviously inhibited proliferation of the three ovarian cancer cell lines, down-regulated the protein levels of CDK4, CDK6, and cyclin Dl, and blocked the cell cycle progressions in G1 phase. Meanwhile, AB23 induced accumulation of the sub-G1 phase in the three cell lines in a concentration dependent manner. The protein levels of cleaved poly ADP-ribose polymerase (PARP) and the ratio of Bax/Bcl2 were up-regulated after treatment with AB23. Further study showed that AB23 induced endoplasmic reticulum stress through 1RE1 signaling pathway and silencing of IRE1[alpha] partially enhanced AB23-induced apoptosis. Wound healing and transwell assays showed that AB23 could also suppress the migration and invasion of HEY cells. Moreover, it down-regulated the protein levels of matrix metalloproteinases MMP-2 and MMP-9.
Conclusion: AB23 possessed anti-proliferation, anti-migration and anti-invasion activities as a single agent on ovarian cancer cells.
Alisol B 23-acetate
Cell cycle arrest
Ovarian cancer is the first leading cause of death among gynecologic malignancies worldwide. According to the 2014 World Cancer Report, approximately 239,000 women were diagnosed with ovarian cancer and 152,000 women died of this disease in 2012 (Stewart and Wild 2014). Approximately 22,280 women will receive a new diagnosis of ovarian cancer in the United States and 14,240 women will die from this disease in 2016 (Siegel et al. 2016). It remains extremely difficult to treat, and the 5 year survival rate of ovarian cancer remains below 40%, unchanged for the past decades (Colombo et al. 2014, Vaughan et al. 2011). Surgery is still the first choice for the treatment of ovarian cancer. However, owing to the difficulty in diagnosing the disease at an early stage, high rates of metastasis and recurrence, surgery alone is not sufficient to control these tumors. Adjuvant radiotherapy, chemotherapy and biological therapy after resection have been clinically adopted to improve the survival status of patients, among which chemotherapy has been recommended as the relatively effective strategy. According to the 2015 National Comprehensive Cancer Network ovarian cancer treatment guidelines, for most patients with stage III-IV ovarian cancer, the standard pladnum-taxane doublet is the first-line chemotherapy, most commonly carboplatin/cisplatin and paclitaxel (Sayal et al. 2015). Although initial chemotherapy is effective in many patients, the relapse rate remains high and tumors become increasingly resistant. Relapse occurs in up to 70% of patients faced with chemoresistance within 5 years. Chemoresistance has become a major cause of treatment failure in human ovarian cancer. Therefore, it is imperative to develop new chemotherapeutic drugs to overcome the intractable chemoresistance.
Discovery of novel anti-cancer compounds from natural products have received more and more attention owing to their rich source and enormous structural diversity (Li et al. 2013, Wu et al. 2013, Xu et al. 2014). Alisol B 23-acetate (AB23) is a protostane-type triterpene isolated from Alismatis Rhizoma which is a medicinal plant widely used in traditional Chinese medicine for urological disease for a long time. Its chemical structure is shown in Fig. 1A. In recently years, the biological characterization of AB23 has been identified and several pharmacological activities have been defined, including hepatoprotective (Meng et al. 2015a, Meng et al. 2015b), anti-hepatitis virus (Jiang et al. 2006), anti-bacterial (Jin et al. 2012), etc. Furthermore, AB23 has been demonstrated to possess anti-proliferative activity in our recent study on the screening of anti-proliferative activities of terpenoids isolated from Alismatis Rhizoma (Xu et al. 2015). It also induced Bax nuclear translocation and apoptotic cell death in human hormone-resistant prostate cancer PC-3 cells (Huang et al. 2006). However, there are still few systematical studies about the effects of AB23 on multiple processes of tumor progression in ovarian cancer cells.
In this study, the effects of AB23 on various ovarian cancer cells were systematically explored, including cell viability, cell cycle, apoptosis, migration and invasion, and the relative signaling pathways. The results showed that AB23 might be a promising lead compound for the treatment of ovarian cancer.
Materials and methods
AB23 was obtained from National Institutes for Food and Drug Control (Beijing, China). It was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Co., St. Louis, MO, USA) as a stock solution and stored at -20[degrees]C. Dulbecco's modified Eagle's medium (DMEM), RPMI1640 medium, fetal bovine serum (FBS), Penicillin-Streptomycin (10,000 units/ml of penicillin and 10,000 [micro]g/ml of streptomycin), 0.25% trypsin-EDTA and phosphate-buffered saline (PBS) were purchased from Gibco (Carlsbad, CA, USA). Paditaxel and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (Saint Louis, MO, USA). Propidium iodide (PI) was purchased from and Invitrogen (Carlsbad, CA, USA). Primary antibodies against PARP, Bd-2, Bax, cydin D1, cyclin-dependent kinase (CDK) 4, CDK6, IRE1[alpha], p-eIF21[alpha], Bip, and GAPDH, together with the secondary antibodies, were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).
The human ovarian cancer cell HEY was kindly provided by Dr. Wen-An Qiang (Northwestern University Feinberg School of Medicine, Chicago, IL, USA), A2780 and paclitaxel-resistant A2780 (A2780/Taxol) cells were acquired from KeyGEN Biotech (Nanjing, Jiangsu, China). HEY and A2780 were cultured in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin, and A2780/Taxol cells were cultured in RPMI1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin. Cells were grown in a standard humidified incubator with 5% C[O.sub.2] at 37 [degrees]C.
The cytotoxicity of AB23 was determined by MTT assay. Exponentially growing A2780, A2780/Taxol and HEY cells were seeded into 96-well plates. Upon reaching approximately 70-80%, the cells were treated with a series of concentrations of AB23 for 24 h. Then, 1 mg/ml MTT solution was added to each well and the 96well plates were further incubated for 4h at 37[degrees]C. To dissolve the needle-like formazan crystals formed by viable cells, 100 [micro]l of DMSO was added to each well and the absorbance at 570 nm was measured using a microplate reader (Perkin Elmer, 1420 Multilabel Counter Victor 3, Wellesley, MA, USA).
Flow cytometry analysis of DNA content
Cells were treated with various concentrations of AB23 for 24 h, trypsinized, washed with cold PBS and harvested by centrifugation. Then, cells were immediately fixed in 70% ethanol and stored overnight at 4[degrees]C. After 30-minute incubation with PI solution (20 [micro]g/ml) in the dark at room temperature, a total of 10,000 cells were subsequently collected and analyzed by flow cytometry (FACS CantoTM, BD Bioscience, Franklin Lakes, NJ, USA).
Western blotting analysis
Cells were lysed in the lysis buffer containing 1% protease inhibitor cocktail and 1% phenylmethane-sulfonylfluoride, and the protein concentrations of the lysates were determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equal quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and then blocked with 5% nonfat milk for 2h at room temperature. The membranes were incubated with specific primary antibodies overnight at 4 [degrees]C and then probed with corresponding secondary antibodies for 1 h at room temperature. Then, specific protein bands were visualized using ECL Select Western blotting detection reagent (GE healthcare, Buckinghamshire, UK). Equal protein loading was verified by probing with anti-GAPDH antibody.
The target sequence for IRE1[alpha] and scrambled siRNA (no silencing, used as the negative control) were synthesized by GenePharma (Shanghai, China). The target sequences are as follows: (silRE1[alpha]1: sense 5'-CUCCCAGCCAUGAGAAAUAIT-3', antisense 5'-UAUUUCUCAUGGCUCGGAGTT-3'; siIRE1[alpha]2: 5'-GGAA GGUGAUGCACAUCAA-3', antisense 5'-UUGAUGUGCAUCACCUUCC3'; negative control: sense 5'-UUCUCCGAACGUGUCACGUTT-3', antisense S'-ACGUGACACGUUCCGAGAATT-S'). A total of 2 x [10.sup.5] cells/well were seeded onto 6-well plates. A2780 cells were seeded for overnight in 6-well plates and then transfected with IRE1[alpha] and scrambled siRNA using lipofectamine 2000 transfection reagent (Invitrogen Corp., Carlsbad, CA, USA), in serum-free medium for 6 h. After further culturing in 10% FBS medium overnight, the cells were treated with AB23 for 24 h and collected for western blotting analysis or Annexin V/PI staining assay.
Annexin V/PI staining assay
Cells were trypsinized, washed with PBS and harvested by centrifugation after treatment. Apoptotic cells were detected using an Annexin V-FITC apoptosis detection kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. Briefly, a total of 10,000 cells were subsequently collected and analyzed by flow cytometry.
Wound healing assay
HEY cell migration was assessed using the wound healing assay. Upon 70-80% of the 24-well plate was covered, two perpendicular lines of cells were scraped away using a 200 [micro]l pipette tip. Then, the medium was changed into fresh serum-free medium with different concentrations of AB23, and three randomly selected fields along the scraped line in each well were photographed using an ++AxioCam HRc CCD camera (Carl Zeiss, Thornwood, NY, USA). After a 24 h incubation, the views of the selected fields were photographed again. The average scraped width per well was measured and subtracted from the measurement prior to treatment. The change in the width for each experimental condition was compared with that of the control group.
The invasive and migration potential of HEY cells were assessed using transwell assay according to the method previously reported with minor modifications (Nizamutdinova et al. 2008). For invasion and migration assay, the upper side of each chamber was coated with 100 [micro]l Matrigel (diluted 1:12 in PBS) or 100 [micro]l PBS and dried for 30 min at 37 [degrees]C. Then, HEY cells were suspended in 0.5% FBS DMEM with different concentrations of AB23 and deposited into the upper chamber per well. The lower chambers were filled with 500 [micro]l complete medium containing relative concentrations of AB23. After 24 h incubation, cells on the upper side of the membranes were removed using cotton swabs, and the invasive cells on the lower surface of the membranes were fixed by 4% paraformaldehyde and stained with crystal violet (Beyotime Biotechnology, Shanghai, China) for 30 min at room temperature. Three randomly selected fields were photographed using the AxioCam HRc CCD camera. The invasive and migration ability of cells was quantified by counting the number of cells that invaded the underside of the porous polycarbonate membrane.
Data were expressed as mean [+ or -] SD. Statistical significances were analyzed by Student's t test and one-way analysis of variance (one-way ANOVA) using SPSS 17 software (Statistical Package for the Social Sciences, SPSS Inc., Chicago, IL, USA). * P < 0.05 and ** P < 0.01 were considered as the significant difference.
AB23 decreased the cell viability of ovarian cancer cells
The human ovarian cancer cell lines A2780, A2780/Taxol and HEY were used to detect the anti-cancer potential of AB23. Firstly, the parental A2780 and paclitaxel-resistant A2780/Taxol cells were assessed by detection of survival rate against paclitaxel. As shown in Fig. IB, A2780/Taxol cells were much less sensitive to paclitaxel compared to the parental A2780 cells. 0.625 [micro]M of paclitaxel showed an inhibitory rate of almost 80% in A2780 cells while 10 [micro]M of paclitaxel just exhibited an inhibitory rate of less than 40% in A2780/Taxol cells, indicating a very high resistance index. Then, cells were treated with various concentrations of AB23 for 24 h and the cell viability was determined by MTT assay. The results showed that AB23 dose-dependently inhibited the cell proliferation in the three ovarian cancer cell lines (Fig. 1C, D, E). Half inhibitory concentrations (I[C.sub.50]) of AB23 in A2780, A2780/Taxol and HEY cells were 11.21 [+ or -] 1.08, 15.18 [+ or -] 1.09 and 10.73 [+ or -] 0.62 [micro]M, respectively (Fig. IF). The resistance index of the paclitaxel-resistant A2780/Taxol and parental A2780 cells to AB23 was 1.35, indicating that AB23 presents anti-multidrug resistance activity.
AB23 induced G1 phase cell cycle arrest in ovarian cancer cells
Analysis of the distribution of cell cycle showed that AB23 effectively induced G1 phase cell cycle arrest in the three ovarian cancer cell lines at low concentrations that didn't induce obvious cell cytotoxicity. Following 24 h exposure to relative concentrations of AB23, the fraction of A2780, A2780/Taxol and HEY cells at G1 phase increased from 47.52 [+ or -]8.29% to 78.93 [+ or -]2.77% (9[micro]M), 51.86 [+ or -]6.42% to 64.87 [+ or -]3.32% (9[micro]M), and 53.58 [+ or -]1.70% to 71.17 [+ or -]7.99% (10[micro]M), respectively (Fig. 2A). To further explore AB23-mediated G1 phase cell cycle arrest, some proteins related to G1 cell cycle proceeding were detected using western blotting. Consistent with the results of the FACS assay, the expression of cyclin D1, CDK4 and CDK6 was down-regulated after 24 h-AB23 treatment (Fig. 2B), indicating that AB23 might induce G1 phase cell cycle arrest by modulating the cyclin-CDKs.
AB23 induced apoptosis in ovarian cancer cells
As induction of apoptosis is one of the major causes that mediate inhibition of cell proliferation, we tested the increase of sub-G1 phase using flow cytometry, which is a characteristic of apoptosis. AB23 induced a dose-dependent increase in sub-G1 phase in the three cell lines, indicating apoptotic phenomena. After treatment with relative high concentrations of AB23 (12 [micro]M in A2780 and A2780/Taxol, 15 [micro]M in HEY), 37.52 [+ or -] 10.54%, 39.58 [+ or -] 14.14%, 47.34 [+ or -] 5.89% of cells underwent sub-G1 phase in A2780, A2780/Taxol and HEY, respectively (Fig. 3A). We further detected the levels of several proteins that related with apoptosis. After AB23 treatment, the cleavage fragment of PARP was obviously up-regulated as well as the Bax/Bcl-2 ratio (Fig. 3B, C), which is consistent with the enhanced apoptosis.
Knockdown of IRE1[alpha] increased AB23-induced apoptosis in ovarian cancer cells
Our previous study has demonstrated that AB23 could induce ER stress in human hepatocellular carcinoma HepG2 cells (Xu et al. 2015). Herein, to further investigate whether AB23 induced ER stress in ovarian cancer cells, we determined the expression levels of several crucial components of ER stress process after AB23 treatment. As shown in Fig. 4A and B, AB23 obviously induced up-regulation of IRE1[alpha] and Bip in a dose- and time-dependent manner while it didn't affect the eIF2a phosphorylation, indicating that AB23 might activate the IRE1 pathway in the ovarian cancer A2780 and HEY cell lines.
Then, the relationship between AB23-induced apoptosis and up-regulation of IRE1[alpha] was further investigated by siRNA-mediated specific knockdown of IRE1[alpha] in A2780 and HEY cells. Compared with the negative control group, IRE1[alpha] was obviously down-regulated by the two siRNA sequences. Silencing of IRE1[alpha] partially up-regulated AB23-induced cleavage fragment of PARP and increased the Annexin V positive cell population, indicating that knockdown of IRE1[alpha] could increase AB23-induced apoptosis in ovarian cancer A2780 (Fig. 5A, B) and HEY (Fig. 5C, D) cells.
AB23 inhibited migration and slightly affected invasion in HEY cells
Migration and invasion of cancer cells are critical steps of ovarian cancer metastasis. HEY was reported to be a relative highly invasive ovarian cancer cell line (Ahmed et al. 2003), and our previous data also indicated A2780 and A2780/Taxol cells to possess relative low ability of migration and invasion, so we further investigated the effect of AB23 on migration and invasion of HEY cells. Wound healing assay and transwell assay (no Matrigel) were conducted to evaluate the inhibitory effect of AB23 on cell migration. The results of wound healing assay showed that 24 and 48 h treatment of AB23 obviously inhibited migration of HEY cells (Fig. 6A). The results of transwell assay (no Matrigel) also indicated that AB23 treatment decreased the migrative cells (Fig. 6B). Matrigel-coated transwell assay was employed to evaluate cell invasion. As shown in Fig. 6C, low concentrations of AB23 slightly affected invasion of HEY cells. Besides, the western blotting analysis indicated that AB23 dose dependently down-regulated the expression levels of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 (Fig. 6D), which have been reported to play a crucial role in tumor cell invasion (Fan et al. 2015).
In our previous screening of anti-proliferative activities of terpenoids isolated from Alismatis Rhizoma, AB23 was demonstrated to possess anti-proliferation activity in hepatocellular carcinoma and human breast adenocarcinoma cells (Xu et al. 2015). Herein, we further found that AB23 obviously induced G1 phase cell cycle arrest in ovarian cancer cells. Together with the data obtained from flow cytometry and western blotting analysis, AB23 indeed induced up-regulation of Bax/Bcl-2 and contribute to the initial of apoptosis. Particularly, AB23 showed remarkable activity on the paditaxel-resistant ovarian cancer A2780 cells, indicating that it might be used for treatment of chemoresistant ovarian cancer. Since many novel compounds with prominent anti-cancer activity also induce serious side effects (Wang et al. 2011, Ye et al. 2010), in vivo safety evaluation is necessary for the development of new drug. Our recent study about the 90-day subchronic oral toxicity in rats has confirmed the safety of the triterpene-enriched extract from Alismatis Rhizoma (Huang et al. 2013). Since AB23 is one of the major components of the extract (Hur et al. 2007), this may support the further investigation and potential therapeutic use of AB23.
ER stress and the subsequent unfolded protein response (UPR) have been widely reported to have especially close relations with cell apoptosis (Chan et al. 2015, Verfaillie et al. 2013, Walter et al. 2015). In mammalian cells, IRE1 and PERK independently govern two key ER stress and UPR signal transduction pathways (Ron and Walter 2007). To explore whether AB23 induced ER stress in ovarian cancer cell line, we detected the proteins levels of IRE1[alpha] and phosphorylation eIF2[alpha], which are of IRE1 and PERK pathways, respectively. It is reported that extended PERK and IRE1 signaling pathways have opposite effects on cell viability, differential activation of PERK and IRE1 pathways may determine life or death decisions after ER stress (Drogat et al. 2007, Lin et al. 2009). Sustained PERK signaling pathway could impair cell proliferation and promote apoptosis while equivalent durations of 1RE1 signaling pathway could enhance cell proliferation without promoting cell death (Lin et al. 2009). Coincidentally, our results indicate that AB23 might activate the IRE1 pathway while didn't affect the PERK pathway. Owing to the cell cycle arrest effect, AB23 didn't induce very high rate of Annexin V positive cells. However, silencing of IRE1[alpha], at least in part, enhanced AB23-induced apoptosis.
Since the poor prognosis of ovarian cancer is largely due to the high rate of metastasis, inhibition of metastasis is a promising stratagem for treatment of ovarian cancer (Naora and Montell 2005, Yeung et al. 2015). Wound healing and transwell assays have been widely used to evaluate cancer metastasis in vitro (Fujimura et al. 2015, Gutschner et al. 2013). Herein, we found that AB23 significantly inhibited HEY cell migration in a concentration-dependent manner after 24 h treatment in the wound healing assay. MMPs have been widely reported to implicate in promoting tumor metastasis (Egeblad and Werb 2002, Kessenbrock et al. 2010). MMP-2 and MMP-9, which can degrade collagen IV, have been suggested to be critical for cancer invasive and metastatic potential (Chen et al. 2012). Our data showed that AB23 downregulated the protein levels of MMP-2 and MMP-9, which contributes to the metastasis inhibition of AB23. This phenomena still deserves further study since MMP-2 and MMP-9 expression levels are regulated at many levels, including gene and proenzyme activation, by numerous oncogene and tumor suppressor pathways (Dong et al. 2013). It is also noteworthy that treatment with 5 [micro]M AB23 reduced the cell viability for about 15% in HEY cells (Fig. IE), and AB23 induced G1 phase cell cycle arrest which could result in proliferation inhibition. Thus, cell cytotoxicity via G1 cell cycle arrest and apoptosis could not be excluded when the concentration of AB23 is high.
In conclusion, AB23 inhibited proliferation of ovarian cancer cells including the paditaxel-resistant one. It could down-regulate the expression levels of G1 cell cycle-related proteins and blocked the cell cycle progressions in G1 phase. Meanwhile, AB23 induced apoptosis and ER stress in the three cell lines, and inhibition of ER stress via silencing of IRE1[alpha] partially enhanced AB23-induced apoptosis. Moreover, AB23 could also suppress the migration and invasion of HEY cells. Our results support AB23 to be a candidate chemotherapeutic agent for ovarian cancer treatment.
Received 14 January 2016
Revised 6 April 2016
Accepted 7 April 2016
Conflict of interest
The authors declare no conflict of interest.
This study was supported by Science and Technology Development Fund, Macao S.A.R (FDCT) (038/2014/A1) and Research Fund of University of Macau (One Time Special Fund for PhD Student in University of Macau, MRG024-LJJ-2015-ICMS, MYRG2015-000911CMS-QRCM, MYRG2015-00101-ICMS-QRCM and CPG2014-00012ICMS).
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Le-Le Zhang (a), Yu-Lian Xu (a), Zheng-Hai Tang (a), Xiao-Huang Xu (a), Xin Chen (a), Ting Li (a), Chun-Yong Ding (b), Ming-Qing Huang (c), Xiu-Ping Chen (a), Yi-Tao Wang (a), Xiao-Feng Yuan (d) **, Jin-Jian Lu (a) *
(a) State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China
(b) State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
(c) College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China
(d) College of Life Science, Zhejiang Chinese Medical University, Hangzhou, China
Abbreviations: AB23, alisol B 23-acetate; DMSO, dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; Pi, propidium iodide; PARP, poly ADP-ribose polymerase; MMP, matrix metalloproteinases; CDK, cyclin-dependent kinase; A2780/Taxol. Paditaxel-resistant A2780; 1C50, half inhibitory concentrations; ER, endoplasmic reticulum; UPR, unfolded protein response.
* Corresponding author at: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao.
** Co-corresponding author.
E-mail addresses: firstname.lastname@example.org (X.-F. Yuan), email@example.com, firstname.lastname@example.org (J.-J. Lu).
Fig. 1. AB23 decreased the cell viability of ovarian cancer cells. (A) Chemical structure of AB23. (B) A2780 and A2780/Taxol cells were treated with different concentrations of paclitaxel for 48 h and the cell viability was tested by MIT assay. (C-E) A2780, A2780/Taxol and HEY cells were treated with series concentrations of AB23 for 24 h and the cell viability was tested by MTT assay. *P< 0.05 and **P< 0.01, compared with the 0 [micro]M AB23 treatment (control). (F) [IC.sub.50] values of AB23 in A2780, A2780/Taxol and HEY cells. F Cell line [IC.sub.50] (mean SD, [micro]M) A2780 11.21 [+ or -] 1.08 A2780/Taxol 15.18 [+ or -] 1.09 HEY 10.73 [+ or -] 0.62
Please note: Some tables or figures were omitted from this article.
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|Author:||Zhang, Le-Le; Xu, Yu-Lian; Tang, Zheng-Hai; Xu, Xiao-Huang; Chen, Xin; Li, Ting; Ding, Chun-Yong; Hu|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jul 15, 2016|
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