Cytotoxic effects of kazinol A derived from Broussonetia papyrifera on human bladder cancer cells, T24 and T24R2.
Background: Broussonetia papyrifera (B. papyrifera), also known as paper mulberry, has been used as a traditional medicine for the treatment of several diseases, including ophthalmic disorders and impotency. However, the biological activity of kazinol A (1) among flavonols isolated from B. papyrifera has not been identified.
Purpose: We identified a candidate metabolite for anti-human bladder cancer treatment from B. papyrifera and investigated the possible molecular mechanisms underlying its cytotoxic effects in T24 and cisplatin-resistant T24R2 human bladder cancer cells.
Methods: T24 and T24R2 cells were treated with five flavonols from B. papyrifera and their cytotoxic effects were determined using MTT assay, cell cycle analysis, mitochondrial membrane potential, and propidium iodide staining. Autophagy rate was calculated by counting LC3-GFP dots in the cells. All related protein expressions were analyzed by immunoblotting.
Results: Compound 1 showed relatively higher cytotoxicity in the human bladder cancer cells, T24 and T24R2, rather than other tissues-originated cancer cells. Compound 1 significantly attenuated cell growth through [G.sub.0/1] arrest mediated by a decrease in cyclin D1 and an increase of p21. Apoptosis and autophagy induced by compound 1 treatment was accompanied by a modulation of the AKT-BAD pathway and AMPK-mTOR pathway, respectively.
Conclusions: Our results suggest that compound 1 induces cytotoxic effects in human bladder cancer cells, including the cisplatin-resistant T24R2. Compound 1 may be a candidate for the development of effective anti-cancer drug on human urinary bladder cancer.
Urinary bladder cancer
Bladder cancer is one of the most common urinary tract cancers and the eleventh most common cancer worldwide. At diagnosis, approximately 30% of bladder cancer patients have muscle invasive cancer cells and 10% of patients have metastatic disease with a poor prognosis (Arantes-Rodrigues et al., 2013). Although the only treatment strategy in these cases is cisplatin-based combination chemotherapy, approximately 30% of patients do not respond to treatment (Fossa et al., 1996; Saxman et al., 1997). Therefore, research to identify agents with increased therapeutic effects is necessary.
Broussonetia papyrifera (B. papyrifera), also known as paper mulberry, grows naturally in Asian and Pacific countries and has been used as a traditional medicine for the treatment of several diseases, including ophthalmic disorders and impotency (Lee et al., 2001), and as a folk medicine in China for the treatment of gynecological bleeding, dropsy, and dysentery disease (Feng et al., 2008). Several types of flavonoids have been identified from the leaves of this plant, and some of them exhibit strong properties, including antiplatelet (Lin et al., 1996), antioxidant, anti-inflammatory (Chen et al., 2002), aromatase inhibitory (Lee et al., 2001), secretory phospholipase A-2 inhibitory (Kwak et al., 2003), PTPIB enzyme inhibitory (Chen et al., 2002), antimicrobial, cytotoxic (Sohn et al., 2004), and tyrosinase inhibitory activities (Zheng et al., 2008).
In this study, we purified five flavonols from B. papyrifera and determined the cytotoxic effects of each in the human bladder cancer cell T24 and its cisplatin-resistant derivative T24R2. We also studied the possible molecular mechanisms how compound 1 among the flavonols induces the cytotoxic effects.
Materials and methods
Cell lines and cultures
SW620, MCF-7, T98GP2, T24 cancer cell lines, and HEK293 cell line were obtained from the American Type Culture Collections (ATCC, VA, USA). The cisplatin-resistant derivative cell line of T24, T24R2, was established by serial desensitization of T24 cells (Hong et al., 2002). Cells were maintained as a monolayer culture in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma, MO, USA) and 1% penicillin/streptomycin (WelGENE, Deagu, Korea) in a humidified chamber with 5% C[O.sub.2] at 37 [degrees]C.
B. papyrifera roots were collected in July 2008, from a hillside (200 m altitude) in the Gyeongnam province in south-east Korea and identified by Prof. Myong Gi Chung. A voucher specimen was deposited at the herbarium of Kyungpook National University (KNU) under the number KHPark 210709.
Extraction and isolation
Extraction and isolation of metabolites from B. papyrifera were performed according to a previous report, with modification (Ryu et al., 2010). Briefly, dried root barks of B. papyrifera (1.0 kg) were powdered and extracted with EtOH at room temperature. The extract was concentrated on a rotatory evaporator and kept in a desiccator for complete removal of the solvent to give a dark brown residue (143g, 14.3%). A portion of this residue (5g) was fractionated on a reversed-phase (RP) silica gel column (5 x 30 cm, YMC ODS AQ-HG 10 [micro]m, 220 g) using MPLC (Puriflash 450, France, Interchim) with a linear gradient of 0%-100% C[H.sub.3]OH/[H.sub.2]O and a flow rate of 30ml/min to obtain nine fractions (BP1-9). The fraction enriched with compound 5 was further purified by silica gel flash CC. The fraction enriched with compound 2 was purified by reversephase CC (ODS-A, 12 nm, S-150 [micro]m), eluting with C[H.sub.3]OH/[H.sub.2]O (4:1) to obtain compound 2. The faction enriched with compound 3 and 4 was subjected to flash CC employing a CH[Cl.sub.3]/acetone gradient (30:1 [right arrow] 5:1) to obtain compounds 3 and 4. The subfraction BP7, enriched with compound 1, was subjected to flash CC using a hexane/ethyl acetate gradient (30:1 -+1:1) to obtain compound 1 (115 mg). Finally, the purified compound 1 was identified by comparing its [sup.1]H and [sup.13]C NMR data (Supplementary Information).
Kazinol A (1): yellowish powder; mp 129-130 [degrees]C; [[[alpha]].sub.D]-10.7 [degrees] (CH[Cl.sub.3], c 0.13); EIMS, m/z 394 [[M].sup.+]; HREIMS, m/z 394.2141 (calculated for [C.sub.25][H.sub.30][O.sub.4], 394.2144); [sup.1]H and [sup.13]C NMR data were consistent with previously published data (Ryu et al., 2010).
Broussochalcone B (2): yellowish powder; mp 168-169 [degrees]C; EIMS, m/z 324 [[M].sup.+]; HREIMS, m/z 324.1363 (calculated for [C.sub.20][H.sub.20][O.sub.4], 324.1362); [sup.1]H and [sup.13]C NMR data were consistent with previously published data (Ryu et al., 2010).
8-(1,1-Dimethylallyl)-5'-(3-methylbut-2-enyl)-3',4',5,7- tetrahydroxyflanvonol (3): amorphous yellow powder; mp 73-74 [degrees]C; EIMS, m/z 438[[M].sup.+]; HREIMS, m/z 438.1648 (calculated for [C.sub.25][H.sub.26][O.sub.7], 438.1679); [sup.1]H and [sup.13]C NMR data were consistent with previously published data (Chen et al., 2002).
Papyriflavonol A (4): amorphous yellow powder; mp 202-204 [degrees]C; EIMS, m/z 438 [[M].sup.+]; HREIMS, m/z 438.1647 (calculated for [C.sub.25][H.sub.26][O.sub.7], 438.1679); [sup.1]H and [sup.13]C NMR data were consistent with previously published data (Zhang et al., 2001).
3'-(3-Methylbut-2-enyl)-3',4'.7-trihydroxyflavane (5): yellow sticky oil; [[[alpha]].sub.D] -5.8 [degrees] (CH[Cl.sub.3], c 0.35); EIMS, m/z 326 [[M].sup.+]; HREIMS, m/z 326.1516 (calculated for [C.sub.20][H.sub.22][O.sub.4], 326.1518); [sup.1]H and [sup.13]C NMR data were consistent with previously published data (Chen et al., 2002).
The cytotoxicity of compound 1 dissolved in DMSO was evaluated by MTT assays performed on T24 and T24R2 cells. Cells were seeded in a 96-well plate (5 x [10.sup.3] cells/well) and then incubated for 24 h in the absence or presence of compound 1. A total of 30 pi MTT solution (5 mg/ml) was added. And then the cells were incubated for another 3 h. After the medium was completely removed, 200 pi of DMSO was added to each well to extract the formazan products formed by viable cells. The absorbance of the solutions was measured on a Bio-Rad 550 microplate reader at 595 nm. The relative cell viability (%) was determined by comparing the absorbance at 595 nm with control, which was treated with DMSO.
Antibodies and reagents
Anti-caspase-3, anti-PARP, anti-cyclin Dl, anti-p21, anti-LC3 II, anti-p-AMPK, anti-AMPK, anti-p-AKT (T308), anti-AKT, anti-pmTOR, anti-mTOR, and anti-pBad (S136) antibodies were purchased from Cell Signaling Technology (MA, USA). The anti-active Bax antibody was purchased from BD (CA, USA). Anti-[alpha]-tubulin and Bad antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Methylthiozolyldi-phenyl-tetrazolium bromide (MTT), propidium iodide (PI), pan caspase inhibitor (Z-VAD-fmk), 3-methyladenine (3-MA), and 4', 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma.
Cells were seeded in six-well plates at a density of 2.4 x [10.sup.5] cells/well followed by treatment with compound 1. After 24 h, the cells were trypsinized, washed with PBS, and collected. The resulting pellet was fixed in 70% ethanol and stored overnight at -20 [degrees]C. The cells were washed with PBS and treated with RNase (Sigma) followed by staining with PI (Sigma). Cells were then subjected to flow cytometric analysis using FACSVerse[TM] (Beckman Coulter Fullerton, CA, USA), and data were analyzed using Flowjo VIO (FlowJo, OH, USA).
Mitochondrial permeabilization assays
Cells were incubated with 250 nM Mitotracker[R] Red CMXRos (Invitrogen, M-7512) for 30 min at 37 [degrees]C. Trypsinized cells were washed with PBS several times followed by fixation with 4% paraformaldehyde. Cells were then subjected to flow cytometric analysis using FACSVerse[TM], and data were analyzed using Flowjo V10.
Total RNA was extracted from cells using a total RNA isolation solution (RiboEx[TM]; GeneAll, Seoul, Korea) according to the manufacturer's instructions. RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc, TN, USA), and cDNA was synthesized with oligo-dT primers and reverse transcriptase (Fermentas, St. Leon-Rot. Germany). Real-time PCR was performed using an SSoFast[TM] EvaCreen Supermix[R] and CFX96[TM] Real-time detection system (Bio-Rad. CA. USA). The following primers were used for amplification: cyclin D1 5'-CCTAAGTTCGGTTCCGATGA-3', of reverse primer 5'-ACGTCAGCCTCCACACTCTT-3' and p21 forward primer 5'-GACACCACTGGAGGGTGACT-3', of reverse primer 5'-GGCGTTTGGAGTGGTAGAAA-3'.
T24 and T24R2 cells were lysed for 30 min on ice in PROPREP[TM] Protein Extraction Solution (Intron biotechnology, Seongnam, Korea). Protein concentration was determined using the Bradford method. Equal amounts of protein were separated on 8%-15% SDS-PAGE gels. After blotting onto PVDF membranes, nonspecific binding sites were blocked for 1 h with 5% skim milk (Difco Laboratories, Surrey, UK) in Tris-buffered saline containing 0.1% Tween 20. Membranes were incubated overnight using primary antibodies; horseradish peroxidase-conjugated goat antimouse IgG (Sigma) or goat anti-rabbit IgG (Sigma) were used as secondary antibodies. Signals were detected using the Bio-RAD ClarityTM Western ECL substrate (Bio-Rad).
Cells were grown on 25 mm round glass coverslips to 60%-70% confluency and were infected with Microtubule-Associated Protein-1 Light Chain 3 (LC3)(II)-EGFP adenovirus followed by treatment with compound 1 for 14 h Cells were fixed in 4% paraformaldehyde, washed several times with PBS, and mounted with mounting medium (Vector laboratory, CA, USA). Spotted dots in 100 random cells per sample were counted with FluoView[TM] FV1000 at a magnification of 100 (Olympus, Tokyo, Japan). For active Bax staining, fixed and permeabilized cells were incubated with anti-active Bax (BD 556467) overnight followed by anti-Mouse-FlTC for 2 h Cells were washed several times with PBS and mounted with mounting medium.
All data were analyzed using unpaired Student's t-test. Results were considered statistically significant if p < 0.05.
Cytotoxic effects of kazinol A (1) in the human bladder cancer cell lines. T24 and T24 R2
The MIT assay was used to determine the cytotoxic effects of 5 flavonols (Fig. 1A) from B. papyrifera on human bladder cancer cells (Fig. IB). Three Flavonols (compounds 1, 2, and 3) showed cytotoxicity in the T24 cell line, but only compound 1 had cytotoxic effects in the cisplatin-resistant T24-derivative cell line, T24R2. This cytotoxic effect was dependent on the concentration of compound 1 in the two cell lines (Fig. 1C); thus, a 20 [micro]m concentration was selected for subsequent experiments. To determine the cytotoxic effects in various tumor cells, 20 [micro]m of compound 1 was used to treat six different human cell lines, including SW620 (human gastric cancer), MCF-7 (human breast cancer), T98G (human glioblasma), T24, T24R2, and HEK293 (Human Embryonic Kidney 293) cell lines. Among the different cell lines, the cytotoxic effects of compound 1 were the most pronounced in the bladder cancer cell lines (Fig. ID and Supplementary Table 1).
Kazinol A (1)-induced cell cycle arrest
To examine the mechanism involved in the inhibitory effects of compound 1 on T24 and T24R2 cells, cells were incubated with 20 pm compound 1 for 14 h, and the distribution of the cell cycle was analyzed by PI staining and flow cytometry (Fig. 2A). Although T24 was more susceptible to compound 1-mediated [G.sub.0/1] arrest than T24R2, T24R2 also showed statistically significant cell cycle arrest. The percentage of T24 and T24R2 cells in the [G.sub.0/1] phase reached 90.4% and 72.9%, respectively, compared with 43.9% and 53.9% of DMSO control groups, respectively. To identify the specific regulatory genes associated with cell cycle arrest, we performed qRT-PCR and immunoblotting. The observed decrease in the cyclin D1 transcript and increase in the p21 transcript were confirmed using qRT-PCR (Fig 2B), and the protein levels of each were determined using immunoblotting (Fig. 2C and D). Taken together, inhibition of tumor cell growth may be the result of cell cycle arrest at [G.sub.0/1], induced by a decrease in cyclin D1 protein levels and an increase in p21 protein levels.
Kazinol A (1)-induced apoptosis
We further examined whether compound 1 could induce apoptosis in human bladder cancer cells. The percentage of cell death was determined through PI staining, flow cytometry analysis, and calculating the sub-[G.sub.0/1] cell population (Fig. 3A). Cells were treated with 20 [micro]m compound 1 for 24 h; the sub-[G.sub.0/1] populations in T24 and T24R2 cells increased from 5.61% and 1.53% to 42.31% and 26.47%, respectively. The cell death showed typical apoptotic phenotypes such as caspase-3 activation and PARP cleavage (Fig. 3B). Cell death was inhibited by the pan-caspase inhibitor, zVAD-fmk (Fig. 3C). To investigate if mitochondrial outer membrane permeabilization (MOMP) was involved in the apoptosis induced by compound 1, cells were treated with compound 1 followed by staining with Mitotracker[R] Red CMXRos. The membrane potential of mitochondria decreased after compound 1 treatment (Fig. 3D). The loss of mitochondrial membrane potential may be due to Bax activation (Fig. 3E). Therefore, we checked the protein levels of the Bcl-2 family. Among the Bcl-2 family of proteins, Bad has been known as a regulator of Bax activation. Although there was no change in total Bad expression level, Bad phosphorylation on S136 was decreased by compound 1 (Fig. 3D). The dephosphorylated Bad forms a heterodimer with Bcl-2 and Bcl-xl, inhibiting the anti-apoptotic functions of the proteins and allowing Bax to trigger mitochondrial depolarization (Masters et al., 2001). Phospho-AKT levels, a kinase for Bad phosphorylation on S136, were also decreased by compound 1 treatment in T24 and T24R2 cells (Fig. 4D). Based on these data, we concluded that compound 1 induced apoptosis through Bax activation, which was mediated by decreased phospho-Bad due to inhibition of AKT.
Kazinol A (1)-induced autophagic cell death
To test whether compound 1 induces autophagy in bladder cancer cells, we transfected the LC3B-GFP gene construct into cells followed by treatment with 20 [micro]m compound 1 for 14 h As shown in Fig. 4A and B, compound 1 effectively induced GFP-dot formation in the cytosol of cells, which indicates the formation of the autophagosome. To confirm the conversion of LC3-I to LC3II through lipidation, which allows LC3 translocation to form autophagic vesicle membranes, LC3B was directly detected using immunoblotting and LC3B-II formation was increased by compound 1 (Fig. 4C). The conversion of LC3B-I to LC3B-II was inhibited by 3MA, which has been used as a specific inhibitor against autophagy. To evaluate how compound 1 induced autophagy of the cells, the phosphorylation status of AMPK and mTOR were determined using immunoblotting. Whereas AMPK phosphorylation was increased, mTOR phosphorylation was decreased in a time-dependent manner (Fig. 4D). To confirm that AMPK modulation was associated with compound 1 -mediated autophagy, the cells were treated with compound C, a specific inhibitor against AMPK, in the presence of compound 1. As expected, compound C inhibited LC3-II formation as well as AMPK phosphorylation induced by compound 1 (Fig. 4E). Taken together, we suggest that compound 1 induces autophagy in human bladder cancer cells through the modulation of the AMPK/mTOR pathway.
B. papyrifera (paper mulberry), a traditional Chinese medicinal herb, has been used to treat various diseases in eastern countries. Our goals were to find out if active compounds from B. papyrifera could be used for the treatment of human bladder cancer, including drug-resistant forms, and to establish a potential rationale for their clinical application. In this study, our results suggest that compound 1 may be active against human bladder cancer. The cytotoxic effects of compound 1 were tested by analyzing cell proliferation, apoptosis, and autophagy. The present study suggests that AKT, a regulator of cell proliferation and survival (Lawlor and Alessi, 2001), and AMPK, a main intracellular energy-sensing molecule (Hardie et al., 2012), are effective kinases in compound 1-mediated anti-bladder cancer activity.
The Bcl-2 family of proteins are well-defined regulators of mitochondria-induced apoptosis (Adams and Cory, 1998). We found that compound 1 induces apoptosis through a break in the mitochondrial membrane and inhibition of phospho-Bad; no changes in the protein levels of Bcl-2 family proteins were detected. Bad is a pro-apoptotic protein that can dimerize with the anti-apoptotic proteins, Bcl-XL and Bcl-2 (Gross et al., 1999). It is well known that Akt phosphorylates the target protein p70s6k1 at Thr389, which in turn phosphorylates Bad at Serl36 (Zhang et al., 2013). Phospho-Bad interacts with the 14-3-3 protein to form heterodimers and the free form of Bcl-2 or Bcl-XL can interact with Bax to inhibit mitochondria-induced apoptosis (Ayllon et al., 2001). In our system, compound 1 significantly led to the reduction of phospho-AKT levels, which in turn can induce a decrease of phospho-Bad, thus, inhibiting the anti-apoptotic proteins, Bcl-2 and BCL-XL Taken together, we concluded that compound 1 may induce the apoptosis of human bladder cancer cells through AKT inhibition, which leads to dephospho-Bad associated with an increase in active Bax.
Although autophagy and apoptosis seem to be interconnected positively or negatively through molecular networks, the induction of autophagic cell death by some anticancer agents has been underlined as a strategy for the development of new cancer treatments (Gozuacik and Kimchi, 2004). To investigate whether compound 1 can induce autophagy in human bladder cancer, LC3-spot formation in cytosol was evaluated. LC3 spotting was significantly increased by compound 1 and lipidation of LC3B (LC3-II), AMPK phosphorylation, and mTOR dephosphorylation were confirmed using immunoblotting. The AMPK/mTORC pathway has been reported as a key regulatory mechanism, sensing energy in cells and inducing autophagy (Alers et al., 2012). The activation of AMPK induces autophagy formation through ULK1 phosphorylation at Ser317, Ser555, and Ser777, which in turn, induces formation of the ULK1/ATG13/FIP200 complex that phosphorylates Beclin-1, triggering autophagy (Russell et al., 2013). Furthermore, AMPK suppresses mTOR signaling through phosphorylating TSC2 and increasing the activity of the TSC1-TSC2 complex (Kimura et al., 2003). On the other hand, mTOR has been reported as a negative regulator of autophagy in response to growth factors and nutrients (Rubinsztein et al., 2007).
Compound 1 also suppressed cell proliferation through the induction of [G.sub.0/1] cell cycle arrest characterized by an increase in the [G.sub.0/1] cell population, a decrease in cyclin D1 expression, and an increase in p21 expression. The overexpression of cyclin D1 has been associated with a number of cancers and their proliferation (Yamamoto et al., 2006), and the cyclin-dependent kinase inhibitor, p21, negatively regulates the cell cycle by inhibiting the activity of cyclin-CDK complexes through direct interaction (Fotedar et al., 1996).
Research has emphasized that more investigation into the benefits or side effects of the use of herbal medicines is needed because of the lack of toxicity information reported in the literature (Ahn et al., 2010). Side effects from herbal medicines may occur due to pharmaceutical interactions (Safarzadeh et al., 2014). In addition, pharmacodynamic herb-drug interactions may influence qualitative activity and the synergistic or antagonizing properties of the medicine. In addition, pharmacokinetic herb-drug interactions may affect the absorption, metabolism, secretion, and toxicity of the herbal medicines. Although many factors must be considered in order to administer herbal medicines, up to 50% of medicines have originated from natural products or their derivatives (Pan et al., 2013). Therefore, efforts to find candidate compounds from natural sources including plants must be accelerated.
From our data, compound 1 exerts a cytotoxic effect against human bladder cancer cells, including cisplatin-resistant cells, by promoting cell cycle arrest, apoptosis, and autophagic cell death. Furthermore, we suggest that AKT and AM PK are critical regulators of compound 1-induced anti-tumor effects, confirming that both are prime cancer targets.
Kazinol A induced cytotoxic effects against the human bladder cancer cells, T24 and T24R2. These cytotoxic effects were due to apoptosis and autophagic cell death through AKT inhibition and AMPK activation, respectively. Our findings suggest that compound 1 may be used as a lead compound in drug development for the treatment of human bladder cancer, including those forms that are cisplatin-resistant.
Received 29 December 2015
Revised 21 July 2016
Accepted 20 August 2016
Conflict of interest
The authors declare no conflicts of interest.
The authors have nothing to disclose.
This study was supported by next generation biogreen21 (SSAC, PJ01107002), Rural Development Administration, Republic of Korea.
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.08.005.
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Soojong Park (a,1), Ahmad Fudhaili (a,1), Sang-Seok Oh (a), Ki Won Lee (a), Hamadi Madhi (a), Dong-Hee Kim (c), Jiyun Yoo (a), Hyung Won Ryu (d), Ki-Hun Park (a), Kwang Dong Kim (a,b), *
(a) Division of Applied Life Science (BK21 Plus), Gyeongsang National University. Jinju. Republic of Korea
(b) PMBBRC, Gyeongsang National University, Jinju, Republic of Korea
(c) Department of Orthopaedic Surgery, School of Medicine, Gyeongsang National University, Jinju, Republic of Korea
(d) Natural Medicine Research Center, KRIBB, 30-Yeongudanji-ro, Ochang-eup, Cheongwon 363-883, Republic of Korea
Abbreviations: Cald, calculated; DAPI, 4', 6-diamidino-2-phenylindole; DMSO, dimethylsulfoxide; EIMS, Electron Ionization Mass Spectrometric; EtOH, Ethanol; HREIMS, high-resolution electron ionization mass spectrometry; MOMP, Mitochondrial outer membrane permeabilization; MPLC, Medium pressure liquid chromatography; MTT, Methylthiozolyldi-phenyl-tetrazolium bromide; NMR. nuclear magnetic resonance; PBS, Phosphate-buffered saline; PI. Propidium iodide; 3-MA, 3-Methyladenine.
* Corresponding author at: Division of Applied Life Science, Gyeongsang National University, 501, Jinju-daero. Jinju, 660-701, Korea. Fax: +82 55 772 1359.
E-mail address: email@example.com (K.D. Kim).
(1) Soojong Park and Ahmad Fudhaili are co-first authors
Please note: Some tables or figures were omitted from this article.
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|Author:||Park, Soojong; Fudhaili, Ahmad; Oh, Sang-Seok; Lee, Ki Won; Madhi, Hamadi; Kim, Dong-Hee; Yoo, Jiyun|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Nov 15, 2016|
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