Table of Contents Author Guidelines Submit a Manuscript
BioMed Research International
Volume 2018, Article ID 3684845, 8 pages
https://doi.org/10.1155/2018/3684845
Research Article

Inhibitory Effects of Compounds and Extracts from Ampelopsis brevipedunculata on IL-6-Induced STAT3 Activation

Immunoregulatory Material Research Center, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsin-gil, Jeongeup-si, Jeonbuk 56212, Republic of Korea

Correspondence should be addressed to Seung Woong Lee; rk.er.bbirk@codwsl and Mun-Chual Rho; rk.er.bbirk@m-ohr

Received 29 December 2017; Accepted 19 March 2018; Published 10 June 2018

Academic Editor: Fabrizio Montecucco

Copyright © 2018 Hyun-Jae Jang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Ampelopsis brevipedunculata (Maxim.) Trautv. (AB), a traditional East Asian medicine, exhibits protective effects against several inflammatory diseases. Our search for an inhibitor of IL-6-induced JAK2/STAT3 activation revealed that AB ethanolic extract (ABE) had a significant inhibitory effect on IL-6-induced STAT3 expression in Hep3B cells. The isolation and purification of an EtOAc-soluble fraction of ABE (ABEA) using reversed-phase high-performance liquid chromatography (RP-HPLC) afforded 17 compounds. The structures of these compounds (1-17) were elucidated based on 1H and 13C nuclear magnetic resonance (NMR) spectroscopy as well as electrospray-ionization mass spectrometry (ESI-MS) data. ABE and ABEA were screened by a luciferase assay using Hep3B cells transfected with the STAT3 reporter gene. ABEA exhibited potent inhibitory effects on IL-6-induced STAT3 expression; moreover, these effects arose from the inhibition of the phosphorylation of the STAT3, JAK2, and ERK proteins in U266 cells. In addition, the compounds isolated from ABEA were measured for their inhibitory effects on IL-6-stimulated STAT3 expression. Of the compounds isolated, betulin showed the greatest inhibitory effects on IL-6-induced STAT3 activation in the luciferase assay (IC50 value: 3.12 μM). Because of its potential for inhibiting STAT3 activation, A. brevipedunculata could be considered a source of compounds of pharmaceutical interest.

1. Introduction

Interleukin 6 (IL-6), which is involved in various physiological functions including neurodevelopment, haematopoiesis, bone metabolism, and immunity [14], is implicated in many inflammatory diseases, such as asthma, arthritis, osteoporosis, diabetes, cancer, ulcerative colitis, and Crohn’s disease, and plays an important role in inflammation and immune diseases [57]. Thus, after the binding of IL-6 to its receptors, such as membrane-bound or soluble receptors (IL-6R or sIL-6R), the IL-6 complex induces the dimerization and phosphorylation of glycoprotein 130 (gp130). Activation of the IL-6 complex leads to a signalling cascade involving the Janus kinases (JAK)/signal transducer and activator of transcription 3 (STAT3). STAT3 is a member of the STAT family of proteins, and STAT3 plays a role in the response to growth factors and cytokines such as IL-6 [8]. STAT3 is phosphorylated by receptor-associated JAK2 to form homo- or heterodimers and then translocates to the nucleus where it acts as a transcription activator [9]. JAK2 provides “instructions” for producing proteins that promote the growth, development, and proliferation of cells and mediates essential signalling events involved in both innate and adaptive immunity. JAK2/STAT activation has been observed in several types of cancer and tumours, including multiple myeloma, leukaemia, prostate cancer, breast cancer, and colon cancer [10, 11]. The JAK2/STAT signalling pathways is thus very important for cell growth and differentiation, and natural products that target JAK2/STAT have potential for the treatment of cancer and tumours [11].

Ampelopsis brevipedunculata (Maxim.) Trautv. (AB) has beneficial effects on human health, including antioxidant, antihypertension, antiviral, antimutagenic, and skin and liver (antihepatotoxic) protective activities [12, 13]. In Asia, AB has been used in traditional medicine to treat several diseases. Although the components of AB, such as oligostilbenes [14] and phenolics [15], have been reported, the phytochemical constituents of AB are not well known. Therefore, the development of new technologies to identify the bioactive compounds in AB will increase the value of this medicinal plant and will present unique challenges and opportunities for further study.

Thus, the objectives of the present study were to (1) measure the inhibitory effects of ABE and ABEA on Il-6-induced STAT3 activity, (2) evaluate the STAT3, JAK2, and ERK protein levels in the ABEA fraction, (3) and isolate and identify the constituents of ABEA using RP-HPLC, NMR, and ESI-MS. This study will provide a better understanding of the relationship between IL-6 activity and the extracts and compounds.

2. Materials and Methods

2.1. General Procedures

1H, 13C, and 2D NMR spectroscopic data were recorded on a JEOL JNM-ECA600 or JEOL JNM-EX400 instrument (JEOL, Tokyo, Japan) using TMS as a reference. Column chromatography was performed with silica gel (Kieselgel 60, 230-400 mesh, Merck, Darmstadt, Germany), and silica gel 60 F254 and RP-18 F254s (Merck) were used for TLC profiling. Medium-pressure liquid chromatography (MPLC) was performed using a Combiflash RF (Teledyne Isco, Lincoln, NE, USA), and preparative HPLC was performed on a Shimadzu LC-6AD (Shimadzu Co., Tokyo, Japan) instrument equipped with an SPD-20A detector using a Phenomenex Luna C18 (250 mm × 21.2 mm, 5 μm, Phenomenex, Torrance, CA, USA) and YMC C18 hydrosphere (250 mm × 20 mm, S-5 μm, YMC Co., Kyoto, Japan).

2.2. Plant Material

AB was purchased from an herbal store in Seoul, Korea, in May 2014. One of the authors (M. -C. Rho) performed the botanical identification, and a voucher specimen (KRIBB-KR2013-1) has been deposited at the laboratory of the Immunoregulatory Material Research Center at the Jeonbuk Branch of the Korea Research Institute of Bioscience and Biotechnology.

2.3. Extraction and Isolation

Dried and pulverized parts of AB vines (9 kg) were extracted at room temperature with 95% EtOH, and the combined extract was evaporated in vacuo to obtain the EtOH extract (337.8 g). The EtOH extract was suspended in H2O (1.5 L) and progressively partitioned with EtOAc (ABE, 130.1 g). ABEA was separated into 38 subfractions (ABE1-ABE38) by silica gel column chromatography with a solvent system comprising a stepwise gradient of CHCl3/MeOH (1:0–0:1, v/v). ABE3 (187.9 mg) was found to contain β-sitosterol (3), and ABE7 was purified by recrystallization from MeOH to afford 4 (105.7 mg). ABE19 (16.7 g) was subjected to silica gel column chromatography using a stepwise solvent system composed of CHCl3 and MeOH (50:1–0:1, v/v) and separated into 13 subfractions (ABE19A–ABE19M). ABE19B (32.1 mg) was rechromatographed by MPLC (silica gel, 40 g, CHCl3/MeOH, 50:1–0:1, v/v) to yield 1 (2.6 mg), and ABE19D (112.0 mg) was separated by MPLC (silica gel, 40 g, hexane/EtOAc, 1:0–0:1, v/v) to yield 2 (3.5 mg). ABE19H (2.9 g) was further purified by MPLC (C18 130 g, 10–100% MeOH), and 16 (4.3 mg) was separated from ABE19H9 by recrystallization. ABE23 (10.8 g) was chromatographed on an MPLC silica column (silica gel 120 g, n-hexane/EtOAc, 5:1–0:1, v/v) to yield 13 subfractions (ABE23A–ABE23M), and ABE23E (335.2 mg) and ABE23L (56.1 mg) were found to contain compounds 10 and 17, respectively. ABE23A (767.1 mg) was purified by MPLC (silica gel, 40 g, CHCl3/MeOH, 50:1–0:1, v/v) and afforded 6 subfractions (ABE23A1–ABE23A6). Compounds 9 (3.1 mg) and 12 (4.3 mg) were isolated from ABE23A4 (283.4 mg) using preparative HPLC (C18, 15% MeOH, 6 mL/min). Compound 5 (157.4 mg) was isolated from ABE23G (875.5 mg) using MPLC (silica gel, 40 g, n-Hexane/EtOAc, 1:0–0:1, v/v). Compound 8 was obtained from ABE25 (2.0 g) using MPLC (silica gel 120 g, CHCl3:MeOH, 1:0–7:3, v/v), and ABE27 (37.6 g) was chromatographed on a silica gel column eluted with CHCl3 and MeOH (1:0–0:1, v/v) to yield 7 (4.5 g). ABE28 (28.1 g) was subjected to silica gel column chromatography (CHCl3/MeOH, 1:0–0:1, v/v). Among the resulting subfractions (ABE28A–ABE28H), ABE28E (3.5 g) was separated by MPLC (C18 130 g, 10–100% MeOH) to yield 9 fractions (ABE28E1–ABE28E9); compound 15 (4.6 mg) was obtained by the recrystallization of ABE28E7. ABE28E2 (537.2 mg) was separated by MPLC (C18 130 g, 10–100% MeOH) to yield 13 subfractions (ABE28E2A–ABE28E2M). Preparative HPLC (YMC C18 hydrosphere, 10–20% CH3CN, 6 mL/min) was used to isolate compounds 11 (1.5 mg), 6 (5.3 mg), and 13 (14.4 mg) from ABE28E2C (63.2 mg), ABE28E2G (37.7 mg), and ABE28E2H (59.2 mg), respectively. ABE32 (8.6 g) was subjected to silica gel column chromatography, eluted with a mixture of CHCl3 and MeOH (1:0–0:1, v/v), and yielded 10 subfractions (ABE32A–ABE32J). Compound 14 (3.5 mg) was isolated by recrystallization of ABE32I (159.8 mg).

2.4. Cell Culture and Viability

Hep3B (ATCC HB-8064) and U266 (ATCC TIB-196™) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI 1640 medium supplemented with 10% foetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. Cells were maintained in exponential growth phase at 37°C in humidified air with 5% CO2. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess cell viability [16]. Cells were added to each well of a 96-well microplate and incubated at 37°C for 24 h. The cytotoxicity of each fraction was screened at concentrations of 1, 5, 10, and 50 μg/mL. After treatment with the EtOAc or water fraction for 24 h, MTT solution was added to each well, and the plate was incubated for 2 h. To stop succinate-tetrazolium reductase activity and solubilize formazan crystals, 200 μL of dimethyl sulfoxide (DMSO) was added to each well and incubated at 37°C for 1 h. The absorbance of each well was measured at 563 nm using an ELISA reader (BioRad, Model 680, Hercules, CA, USA). Cell viability was expressed as a percentage of the cell viability of the control cells.

2.5. pSTAT3 Luciferase Assay

Human hepatoma Hep3B cells (ATCC HB-8064) stably expressing pSTAT3-Luc were established as described previously [17]. In brief, Hep3B cells expressing pSTAT3-luciferase were seeded in a 96-well culture plate at 2 × 104 cells/well, and the cells were treated with samples for 1 h before stimulation with IL-6 (10 ng/mL) for 12 h. Luciferase activity was measured according to the manufacturer’s protocol (Promega Corp., Madison, WI, USA).

2.6. Western Blotting

Cell proteins were separated by centrifuging the cell lysate for 20 min at 13,000 × g. Equivalent amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes using an electroblotting apparatus. The blots were then immersed in freshly prepared 5% skim milk for a blocking step at room temperature (1 h). Next, the membranes were incubated overnight at 4°C with primary antibody, including anti-phospho-STAT3 (1:1000), anti-STAT3 (1:1000), anti-phospho-JAK2 (1:1000), anti-JAK2 (1:1000), anti-phospho-ERK (1:1000), and anti-ERK (1:1000) antibodies (Cell Signaling, Beverly, MA, USA). The membranes were then incubated with an appropriate horseradish peroxide-conjugated secondary antibody (1:5000) at room temperature. The optical densities of the antibody-specific bands were analysed using a Luminescent Image Analyzer, LAS-3000 (Fuji, Tokyo, Japan) [18].

2.7. Statistical Analysis

All experimental data were obtained in triplicate assays. Data are expressed as the means ± standard deviation (SD). Statistical analyses were performed using Student’s t-test for paired data. Statistical analyses were performed using Prism 5 software (GraphPad software, San Diego, CA, USA). Student’s t-test was used to evaluate the data, and differences were considered statistically significant at P < 0.05 for the mean values of triplicate samples.

3. Results

3.1. Preparation of Extracts and Determination of STAT3 Inhibition

To examine the cytotoxic effects of ABE, ABEA, and water-soluble fractions from ABE (ABW) against Hep3B cells, MTT assays were performed. Cells were treated with various concentrations of these fractions (0, 5, 10, 25, and 50 μg/mL) for 24 h. The cell viability was not affected by ABE, ABEA, or ABW at 50 μg/mL (Figure 1), but at this concentration, the extracts and fractions significantly inhibited IL-6-induced STAT3 expression in Hep3B cells. Notably, ABE and ABEA exhibited stronger inhibitory effects than ABW in a luciferase assay (Figure 1). These data demonstrated that the nonpolar components in ABEA are more potent inhibitors of IL-6-induced STAT3 activity than the polar components of ABW.

Figure 1: The viability of Hep3B cells after treatment with the ethanol (ABE), ethyl acetate (ABEA), and water fractions (ABW) obtained from A. brevipedunculata (a). The inhibitory effects of ABE, ABEA, and ABW on IL-6/STAT3 activation in Hep3B cells were determined using a luciferase assay (b). Genistein was used as a positive control. Values are the means ± SE; p < 0.05, compared with the IL-6-treated group.
3.2. Inhibitory Effects of ABEA on IL-6-Induced JAK2/STAT3 Phosphorylation

To characterize the molecular mechanisms by which ABEA inhibits JAK2, STAT3, and ERK activity, we examined various transcription-related proteins. Western blot analysis demonstrated that the IL-6-induced phosphorylation of STAT3 and JAK2 was reduced in U266 cells after pretreatment with ABEA at 3, 6, and 10 μg/mL (Figures 2(a) and 2(b)). Although a reduction in the pERK/ERK ratio was observed after treatment with ABEA, no significant dose-dependent differences were observed compared to JAK2/STAT3 activation in IL-6-induced U266 cells (Figure 2(c)).

Figure 2: Inhibitory effects of JAK2, STAT3, and ERK activation by ABEA on IL-6-stimulated U266 cell lines. Cells were preincubated with ABEA for 1 h before stimulation with IL-6 (10 ng/mL). Western blot analysis indicated that ABEA inhibited the phosphorylation of JAK2, STAT3, and ERK in U266 cells. The total amount of the corresponding nonphosphorylated protein was used as a loading control for the phosphorylated proteins. Genistein was used as a positive control. Values are the means ± SE; p < 0.05 compared with the IL-6-treated group.
3.3. Isolation of Active Compounds

Bioactivity-guided fractionation and column chromatography purification (Figure 3) of ABEA led to the isolation of compounds 1-17 (Figure 4). The compounds were identified as betulin (1) [19], betulinic acid (2) [20], β-sitosterol (3) [21], β-sitosterol glucoside (4) [22], dihydrokaempferol (5) [23], dihydrokaempferol 3-O-glycoside (6) [24], catechin (7) [25], gallic acid (8) [26], vanillic acid (9) [27], ethyl gallate (10) [28], ethyl gallate 4-O-β-D-glucopyranoside (11) [29], syringic acid (12) [30], benzyl 6′-O-galloyl-β-D-glucopyranoside (13) [31], ellagic acid (14) [32], 3′-O-methylellagic acid 4-O-α-L-rhamnopyranoside (15) [33], 3,3′4′-O-tri-methylellagic acid 4-O-β-D-glucopyranoside (16) [32], and resveratrol (17) [34] by comparison with the spectroscopic data in the literature (Supplementary Material, Figure S1-51). Compounds 3, 4, 6, 11, 13, and 14-16 were isolated from this plant for the first time, and compounds 1, 2, 5, 8, 10-14, and 17 exhibited half-maximal inhibitory concentration (IC50) values ranging from 3.1 to 49.7 μM against IL-6-induced STAT3 activation (Table 1); additionally, with the exception of betulinic acid (2), these compounds showed no cytotoxicity at their IC50 concentrations (Supplementary Material, Figure S52). Betulin (1) exhibited the greatest inhibitory effects on IL-6-induced STAT3 activation with an IC50 value of 3.12 μM.

Table 1: Inhibitory effects of the major compounds from Ampelopsis brevipedunculata on IL-6/STAT3 activation.
Figure 3: Schematic diagram for the isolation of compounds 1-17 from A. brevipedunculata.
Figure 4: Chemical structures of compounds 1-17 from A. brevipedunculata.

4. Discussion

AB has been reported to have various pharmacological activities, such as skin and liver protection and antioxidant activities [12, 35]. In 2014, the anti-inflammatory activity of ABW via inhibition of the PKC-mediated JNK/NF-κB signalling pathways was reported [36]; however, the inhibitory activity of ABEA on IL-6-mediated diseases, such as inflammation, cancer cachexia, rheumatoid arthritis, hypercalcaemia, and multiple myeloma [37], through JAK2/STAT3 signal transduction has not been reported.

IL-6 is a multifunctional cytokine that plays major roles in host defence and immune reactions [38]. Increased levels of IL-6 have been reported in various pathological conditions, such as infection (bacteria and virus), immune disease, inflammation, and tumours [39]. Therefore, regulation of IL-6 function might be effective against various diseases. IL-6 binds to homo- or heterodimer membrane receptor complexes containing glycoprotein 130 (gp130) or the leukaemia inhibitory factor receptor (LIFR) and activates the JAK family of tyrosine kinases [40]. In particular, phosphorylated JAK2 recruits Grb2/Shc/Ras/MEK1/2 and then activates ERK1/2 independently of SHP2 [41]. In Figure 2(c), ABEA suppresses IL-6-mediated ERK phosphorylation; however, no significant differences were observed for at concentrations ranging from 3 to 10 μM compared to the JAK2/STAT3 inhibitory activation. Accordingly, significant differences in the ERK inhibitory activity of ABEA can be observed at concentrations higher than 10 μM, and the main signalling transduction of the IL-6 cascade pathway affected by ABEA is the JAK2/STAT3 signalling pathway.

Except for some phenolics and triterpenes, the chemical constituents of AB are not well known, and our results may provide useful information on the phytochemical properties of AB. We investigated the components of AB using repeated column chromatography. Among the compounds isolated from ABEA, 1, 2, 5, 8, 10-14, and 17 exert inhibitory effects on IL-6-stimulated STAT3 expression, and betulin (1) exhibited the greatest inhibitory activity (Table 1). Betulin, a lupane-type pentacyclic triterpenoid, has various biological activities, such as anti-HIV, anticancer, and anti-inflammatory effects [42, 43], and is a major component in the bark of birch trees such as Betula pendula, B. papyrifera, and B. neoalaskana, which were traditionally used to treat eczema, psoriasis, inflammation, rheumatism, and arthritic diseases [4447].

Based on the present data, we isolated and identified 17 compounds from ABEA, and those compounds were tested for their ability to inhibit IL-6/STAT3 activation using a luciferase reporter assay. In addition, the ABEA fraction was presumed to be involved in inhibiting the expression of genes such as those for STAT3, JAK2, and ERK, which are involved in various signalling pathways. Consequently, the extracts and constituents of AB displayed potent natural inhibition of IL-6/STAT3 in an in vitro model. Overall, the extracts and components have the potential to be developed into new biomaterials. However, further studies are needed to elucidate the precise mechanism of in vivo activity.

Conflicts of Interest

The authors have declared that there are no conflicts of interest.

Authors’ Contributions

Hyun-Jae Jang and Seung-Jae Lee contributed equally to the article as first authors. Hyun-Jae Jang and Seung-Jae Lee designed and carried out the experiments and wrote the manuscript. Hyung-Jin Lim, Kyungsook Jung, and Soyoung Lee performed additional in vitro experiments. Chan Sun Park contributed reagents, materials, and analytical tools. Seung Woong Lee and Mun-Chual Rho discussed and supervised the overall project. All authors read and approved the final manuscript.

Acknowledgments

This research was supported by a grant from KRIBB Research Initiative Program, KGM2221844 and KGS9831815 (2018).

Supplementary Materials

Figure S1: ESI-MS spectrum of 1. Figure S2: 1H NMR (400 MHz, chloroform) spectrum of 1. Figure S3: 13C NMR (100 MHz, chloroform) spectrum of 1. Figure S4: ESI-MS spectrum of 2. Figure S5: 1H NMR (600 MHz, pyridine) spectrum of 2. Figure S6: 13C NMR (150 MHz, pyridine) spectrum of 2. Figure S7: GC-MS spectrum of 3. Figure S8: 1H NMR (400 MHz, chloroform) spectrum of 3. Figure S9: 13C NMR (100 MHz, chloroform) spectrum of 3. Figure S10: FABMS spectrum of 4. Figure S11: 1H NMR (600 MHz, pyridine) spectrum of 4. Figure S12: 13C NMR (150 MHz, pyridine) spectrum of 4. Figure S13: ESI-MS spectrum of 5. Figure S14: 1H NMR (400 MHz, methanol) spectrum of 5. Figure S15: 13C NMR (100 MHz, methanol) spectrum of 5. Figure S16: ESI-MS spectrum of 6. Figure S17: 1H NMR (400 MHz, methanol) spectrum of 6. Figure S18: 13C NMR (100 MHz, methanol) spectrum of 6. Figure S19: ESI-MS spectrum of 7. Figure S20: 1H NMR (400 MHz, methanol) spectrum of 7. Figure S21: 13C NMR (100 MHz, methanol) spectrum of 7. Figure S22: ESI-MS spectrum of 8. Figure S23: 1H NMR (400 MHz, methanol) spectrum of 8. Figure S24: 13C NMR (100 MHz, methanol) spectrum of 8. Figure S25: ESI-MS spectrum of 9. Figure S26: 1H NMR (600 MHz, methanol) spectrum of 9. Figure S27: 13C NMR (150 MHz, methanol) spectrum of 9. Figure S28: ESI-MS spectrum of 10. Figure S29: 1H NMR (400 MHz, methanol) spectrum of 10. Figure S30: 13C NMR (100 MHz, methanol) spectrum of 10. Figure S31: ESI-MS spectrum of 11. Figure S32: 1H NMR (400 MHz, methanol) spectrum of 11. Figure S33: 13C NMR (100 MHz, methanol) spectrum of 11. Figure S34: ESI-MS spectrum of 12. Figure S35: 1H NMR (600 MHz, methanol) spectrum of 12. Figure S36: 13C NMR (150 MHz, methanol) spectrum of 12. Figure S37: ESI-MS spectrum of 13. Figure S38: 1H NMR (400 MHz, methanol) spectrum of 13. Figure S39: 13C NMR (100 MHz, methanol) spectrum of 13. Figure S40: ESI-MS spectrum of 14. Figure S41: 1H NMR (600 MHz, DMSO) spectrum of 14. Figure S42: 13C NMR (100 MHz, DMSO) spectrum of 14. Figure S43: ESI-MS spectrum of 15. Figure S44: 1H NMR (600 MHz, DMSO) spectrum of 15. Figure S45: 13C NMR (150 MHz, DMSO) spectrum of 15. Figure S46: ESI-MS spectrum of 16. Figure S47: 1H NMR (600 MHz, DMSO) spectrum of 16. Figure S48: 13C NMR (150 MHz, DMSO) spectrum of 16. Figure S49: ESI-MS spectrum of 17. Figure S50: 1H NMR (400 MHz, methanol) spectrum of 17. Figure S51: 13C NMR (100 MHz, methanol) spectrum of 17. Figure S52: Cytotoxicity of compounds 1-17 on Hep3B-STAT3-Luc cell lines. (Supplementary Materials)

References

  1. T. Hama, M. Miyamoto, H. Tsukui, C. Nishio, and H. Hatanaka, “Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats,” Neuroscience Letters, vol. 104, no. 3, pp. 340–344, 1989. View at Publisher · View at Google Scholar · View at Scopus
  2. F. Q. Pirih, M. N. Michalski, S. W. Cho et al., “Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6,” PLoS ONE, vol. 5, no. 10, Article ID e13657, 2010. View at Publisher · View at Google Scholar · View at Scopus
  3. N. Kurihara, D. Bertolini, T. Suda, Y. Akiyama, and G. D. Roodman, “IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release,” The Journal of Immunology, vol. 144, no. 11, pp. 4226–4230, 1990. View at Google Scholar · View at Scopus
  4. C. Uyttenhove, P. G. Coulie, and J. Van Snick, “T cell growth and differentiation induced by interleukin-HP1/IL-6, the murine hybridoma/plasmacytoma growth factor,” The Journal of Experimental Medicine, vol. 167, no. 4, pp. 1417–1427, 1988. View at Publisher · View at Google Scholar · View at Scopus
  5. M. F. Neurath and S. Finotto, “IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer,” Cytokine & Growth Factor Reviews, vol. 22, no. 2, pp. 83–89, 2011. View at Publisher · View at Google Scholar · View at Scopus
  6. F. J. Hughes and G. L. Howells, “Interleukin-6 inhibits bone formation in vitro,” Bone and Mineral, vol. 21, no. 1, pp. 21–28, 1993. View at Publisher · View at Google Scholar · View at Scopus
  7. H. Yu, D. Pardoll, and R. Jove, “STATs in cancer inflammation and immunity: a leading role for STAT3,” Nature Reviews Cancer, vol. 9, no. 11, pp. 798–809, 2009. View at Publisher · View at Google Scholar · View at Scopus
  8. J. L. Geiger, J. R. Grandis, and J. E. Bauman, “The STAT3 pathway as a therapeutic target in head and neck cancer: Barriers and innovations,” Oral Oncology, vol. 56, pp. 84–92, 2016. View at Publisher · View at Google Scholar · View at Scopus
  9. S. V. Kuchipudi, “The Complex Role of STAT3 in Viral Infections,” Journal of Immunology Research, vol. 2015, Article ID 272359, 2015. View at Publisher · View at Google Scholar · View at Scopus
  10. M. Kortylewski, R. Jove, and H. Yu, “Targeting STAT3 affects melanoma on multiple fronts,” Cancer and Metastasis Reviews, vol. 24, no. 2, pp. 315–327, 2005. View at Publisher · View at Google Scholar · View at Scopus
  11. A. Xiong, Z. Yang, Y. Shen, J. Zhou, and Q. Shen, “Transcription factor STAT3 as a novel molecular target for cancer prevention,” Cancers, vol. 6, no. 2, pp. 926–957, 2014. View at Publisher · View at Google Scholar · View at Scopus
  12. T. J. Rhim and M. Y. Choi, “The antioxidative effects of Ampelopsisbreipedunculata extract,” Korean Journal of Plant Resources, vol. 23, pp. 445–450, 2010. View at Google Scholar
  13. N. Yabe, K. Tanaka, and H. Matsui, “An ethanol-extract of Ampelopsis brevipedunculata (Vitaceae) berries decreases ferrous iron-stimulated hepatocyte injury in culture,” Journal of Ethnopharmacology, vol. 59, no. 3, pp. 147–159, 1998. View at Publisher · View at Google Scholar · View at Scopus
  14. Y. Oshima and Y. Ueno, “Ampelopsins E, E, H and cis-ampelopsin E, oligostilbenes from Ampelopsis brevipedunculata var. Hancei roots,” Phytochemistry, vol. 33, no. 1, pp. 179–182, 1993. View at Publisher · View at Google Scholar · View at Scopus
  15. A. Inada, Y. Nakamura, M. Konishi, H. Murata, F. Kitamura, and H. Toya, “A New Ionone Glucoside and a New Phenylpropanoid Rhamnoside from Stems of Ampelopsis brevipedunculata (maxim): Trautv,” Chemical & Pharmaceutical Bulletin, vol. 39, no. 9, pp. 2437–2439, 1991. View at Publisher · View at Google Scholar · View at Scopus
  16. H.-J. Kwon, Y.-S. Kim, J.-W. Hwang et al., “Isolation and identification of an anticancer compound from the bark of Acer tegmentosum Maxim,” Process Biochemistry, vol. 49, no. 6, pp. 1032–1039, 2014. View at Publisher · View at Google Scholar · View at Scopus
  17. J. S. Chang, S. W. Lee, M. S. Kim et al., “Manassantin A and B from Saururus chinensis inhibit interleukin-6 - Induced signal transducer and activator of transcription 3 activation in Hep3B cells,” Journal of Pharmacological Sciences, vol. 115, no. 1, pp. 84–88, 2011. View at Publisher · View at Google Scholar · View at Scopus
  18. S.-J. Lee, Y.-S. Kim, J.-W. Hwang et al., “Purification and characterization of a novel antioxidative peptide from duck skin by-products that protects liver against oxidative damage,” Food Research International, vol. 49, no. 1, pp. 285–295, 2012. View at Publisher · View at Google Scholar · View at Scopus
  19. A. Patra, S. K. Chaudhuri, and S. K. Panda, “Betulin-3-caffeate from quercus suber. 13c-nmr spectra of some lupenes,” Journal of Natural Products, vol. 51, no. 2, pp. 217–220, 1988. View at Publisher · View at Google Scholar · View at Scopus
  20. R. H. Cichewicz and S. A. Kouzi, “Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection,” Medicinal Research Reviews, vol. 24, no. 1, pp. 90–114, 2004. View at Publisher · View at Google Scholar · View at Scopus
  21. W.-H. Hui, M.-M. Li, and K.-M. Wong, “A new compound, 21α-hydroxyfriedel-4(23)-en-3-one and other triterpenoids from Phyllanthus reticulatus,” Phytochemistry, vol. 15, no. 5, pp. 797-798, 1976. View at Publisher · View at Google Scholar · View at Scopus
  22. M. Khatun, M. Billah, and M. A. Quader, “Sterols and sterol glucoside from Phyllanthusspecies,” Dhaka University Journal of Science, vol. 60, no. 1, pp. 5–10, 2012. View at Google Scholar
  23. M. Neacsu, P. C. Eklund, R. E. Sjöholm et al., “Antioxidant flavonoids from knotwood of Jack pine and European aspen,” Holz als Roh- und Werkstoff, vol. 65, no. 1, pp. 1–6, 2007. View at Publisher · View at Google Scholar
  24. J. Kang, C. Xie, Z. Li et al., “Flavonoids from acai (Euterpe oleracea Mart.) pulp and their antioxidant and anti-inflammatory activities,” Food Chemistry, vol. 128, no. 1, pp. 152–157, 2011. View at Publisher · View at Google Scholar · View at Scopus
  25. A. L. Davis, Y. Cai, A. P. Davies, and J. R. Lewis, “1H and 13C NMR assignments of some green tea polyphenols,” Magnetic Resonance in Chemistry, vol. 34, no. 11, pp. 887–890, 1996. View at Publisher · View at Google Scholar · View at Scopus
  26. D.-M. Nguyen, D.-J. Seo, and H.-B. Lee, “Antifungal activity of gallic acid purified from Terminalia nigrovenulosa bark against Fusarium solani,” Microbial Pathogenesis, vol. 56, pp. 8–15, 2013. View at Publisher · View at Google Scholar · View at Scopus
  27. J. Y. Cho, J. H. Moon, and K. H. Park, “Isolation and identification of 3-methoxy-4-hydroxybenzoic acid and 3-methoxy-4-hydroxycinnamic acid from hot water extracts of Hoveniadulcis Thunb and confirmation of their antioxidative and antimicrobial activity,” Korean Journal of Food Science and Technology, vol. 32, pp. 1403–1408, 2000. View at Google Scholar
  28. M. Yu, Z. L. Li, and N. Li, “Chemical constituents of the aerial parts of Polygonum capitatum,” Journal of Shenyang Pharmaceutical University, vol. 25, pp. 633–635, 2008. View at Google Scholar
  29. K. De Winter, T. Desmet, T. Devlamynck et al., “Biphasic catalysis with disaccharide phosphorylases: Chemoenzymatic synthesis of α- D -glucosides using sucrose phosphorylase,” Organic Process Research & Development, vol. 18, no. 6, pp. 781–787, 2014. View at Publisher · View at Google Scholar · View at Scopus
  30. S. Inoshiri, M. Sasaki, H. Kohda, H. Otsuka, and K. Yamasaki, “Aromatic glycosides from Berchemia racemosa,” Phytochemistry, vol. 26, no. 10, pp. 2811–2814, 1987. View at Publisher · View at Google Scholar · View at Scopus
  31. J. H. Isaza, H. Ito, and T. Yoshida, “A flavonol glycoside-lignan ester and accompanying acylated glucosides from Monochaetum multiflorum,” Phytochemistry, vol. 58, no. 2, pp. 321–327, 2001. View at Publisher · View at Google Scholar · View at Scopus
  32. D. D. Khac, S. Tran-Van, A. M. Campos, J.-Y. Lallemand, and M. Fetizon, “Ellagic compounds from Diplopanax stachyanthus,” Phytochemistry, vol. 29, no. 1, pp. 251–256, 1990. View at Publisher · View at Google Scholar · View at Scopus
  33. Y. Yazaki and W. E. Hillis, “Polyphenols of Eucalyptus globulus, E. Regnans and E. Deglupta,” Phytochemistry, vol. 15, no. 7, pp. 1180–1182, 1976. View at Publisher · View at Google Scholar · View at Scopus
  34. F. Mattivi, F. Reniero, and S. Korhammer, “Isolation, Characterization, and Evolution in Red Wine Vinification of Resveratrol Monomers,” Journal of Agricultural and Food Chemistry, vol. 43, no. 7, pp. 1820–1823, 1995. View at Publisher · View at Google Scholar · View at Scopus
  35. N. Yabe and H. Matsui, “Ampelopsis brevipedunculata (Vitaceae) extract stimulates collagen synthesis through superoxide generation in the serum-free cultures of rat dermal fibroblasts and Ito cells,” Journal of Ethnopharmacology, vol. 56, no. 1, pp. 67–76, 1997. View at Publisher · View at Google Scholar · View at Scopus
  36. M. Q. Le, M. S. Kim, Y. S. Song, W. N. Noh, S. C. Chun, and D. Y. Yoon, “The water-extracted Ampelopsis brevipedunculata downregulates IL-1β, CCL5, and COX-2 expression via inhibition of PKC-mediated JNK/NF-κB signaling pathways in human monocytic cells,” Journal of Pharmacological Sciences, vol. 126, no. 4, pp. 359–369, 2014. View at Publisher · View at Google Scholar · View at Scopus
  37. A. Enomoto, M.-C. Rho, A. Fukami, O. Hiraku, K. Komiyama, and M. Hayashi, “Suppression of cancer cachexia by 20S,21-epoxy-resibufogenin-3-acetate - A novel nonpeptide IL-6 receptor antagonist,” Biochemical and Biophysical Research Communications, vol. 323, no. 3, pp. 1096–1102, 2004. View at Publisher · View at Google Scholar · View at Scopus
  38. T. Hirano, “Interleukin 6 and its receptor: ten years later,” International Reviews of Immunology, vol. 16, no. 3-4, pp. 249–284, 1998. View at Publisher · View at Google Scholar · View at Scopus
  39. T. Taga and T. Kishimoto, “Gp130 and the interleukin-6 family of cytokines,” Annual Review of Immunology, vol. 15, pp. 797–819, 1997. View at Publisher · View at Google Scholar · View at Scopus
  40. P. C. Heinrich, I. Behrmann, S. Haan, H. M. Hermanns, G. Müller-Newen, and F. Schaper, “Principles of interleukin (IL)-6-type cytokine signalling and its regulation,” Biochemical Journal, vol. 374, part 1, pp. 1–20, 2003. View at Publisher · View at Google Scholar · View at Scopus
  41. A. Fahmi, N. Smart, A. Punn, R. Jabr, M. Marber, and R. Heads, “P42/p44-MAPK and PI3K are sufficient for IL-6 family cytokines/gp130 to signal to hypertrophy and survival in cardiomyocytes in the absence of JAK/STAT activation,” Cellular Signalling, vol. 25, no. 4, pp. 898–909, 2013. View at Publisher · View at Google Scholar · View at Scopus
  42. S. Alakurtti, T. Mäkelä, S. Koskimies, and J. Yli-Kauhaluoma, “Pharmacological properties of the ubiquitous natural product betulin,” European Journal of Pharmaceutical Sciences, vol. 29, no. 1, pp. 1–13, 2006. View at Publisher · View at Google Scholar · View at Scopus
  43. V. Zuco, R. Supino, S. C. Righetti et al., “Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells,” Cancer Letters, vol. 175, no. 1, pp. 17–25, 2002. View at Publisher · View at Google Scholar · View at Scopus
  44. C. A. Dehelean, C. Şoica, I. Ledeţi et al., “Study of the betulin enriched birch bark extracts effects on human carcinoma cells and ear inflammation,” Chemistry Central Journal, vol. 6, no. 1, article 137, 2012. View at Publisher · View at Google Scholar · View at Scopus
  45. P. A. Krasutsky, “Birch bark research and development,” Natural Product Reports, vol. 23, no. 6, pp. 919–942, 2006. View at Publisher · View at Google Scholar · View at Scopus
  46. K. Wacker, C. Gründemann, Y. Kern et al., “Inhibition of corneal inflammation following keratoplasty by birch leaf extract,” Experimental Eye Research, vol. 97, no. 1, pp. 24–30, 2012. View at Publisher · View at Google Scholar · View at Scopus
  47. X. Ci, J. Zhou, H. Lv, Q. Yu, L. Peng, and S. Hua, “Betulin exhibits anti-inflammatory activity in LPS-stimulated macrophages and endotoxin-shocked mice through an AMPK/AKT/Nrf2-dependent mechanism,” Cell Death Disease, vol. 8, no. 5, Article ID e2798, 2017. View at Publisher · View at Google Scholar