Amentoflavone Research Articles

Ultrasonic-Assisted Ionic Liquid Extraction of Two Biflavonoids from Selaginella tamariscina.

Selaginella tamariscina, a traditional Chinese medicine, contains a variety of bioactive components, among which biflavonoids are the main active ingredients and have antioxidant, antitumor, and anti-inflammatory properties. In this study, ultrasonic-assisted ionic liquid extraction (UAILE) is used for the first time to extract two main biflavonoids (amentoflavone (AME) and hinokiflavone (HIN)) from S. tamariscina. A high-performance liquid chromatography method is used for the simultaneous determination of AME and HIN in S. tamariscina. Then, three novel ILs are synthesized for the first time by a one-step method using benzoxazole and three acids or acid salts as raw materials, and the structures of the synthesized ILs are characterized by elemental analysis, infrared spectroscopy, and NMR spectroscopy, as well as the thermal stability of the ILs is evaluated by thermogravimetric analysis. After screening the extraction effects of three benzoxazole ILs, three pyridine ILs, and three imidazole ILs, it is found that [Bpy]BF4 is the best and therefore selected as the extractant. The optimal extraction process is explored in terms of the yields of AME and HIN from S. tamariscina by a single-factor experiments and response surface analysis. Under the optimal level of each influencing factor (IL concentration of 0.15 mol/L, solid–liquid ratio of 1:12 g/mL, ultrasonic power of 280 W, ultrasonic time of 30 min, and three extraction cycles), the extraction rates of AME and HIN from S. tamariscina are 13.51 and 6.74 mg/g, respectively. Moreover, the recovery experiment of [Bpy]BF4 on the extraction of biflavonoids shows that the recovered IL can repeatedly extract targets six times and the extraction rate is about 90%, which indicates that the IL can be effectively reused. UAILE can effectively and selectively extract AME and HIN, laying the foundation for the application of S. tamariscina.

Amentoflavone triggers cell cycle G2/M arrest by interfering with microtubule dynamics and inducing DNA damage in SKOV3 cells.

Ovarian cancer is the seventh most common cancer and the second most common cause of cancer-associated mortality among gynecological malignancies worldwide. The combination of antimitotic agents, such as taxanes, and the DNA-damaging agents, such as platinum compounds, is the standard treatment for ovarian cancer. However, due to chemoresistance, development of novel therapeutic strategies for the treatment of ovarian cancer remains critical. Amentoflavone (AMF) is a biflavonoid derived from the extracts of Selaginella tamariscina, which has been used as a Chinese herb for thousands of years. A previous study demonstrated that AMF inhibits angiogenesis of endothelial cells and induces apoptosis in hypertrophic scar fibroblasts. In order to check the influence of AMF on cell proliferation, the effects of AMF on cell cycle and DNA damage were measured by cell viability, flow cytometry, immunofluorescence and western blotting assays in SKOV3 cells, an ovarian cell line. In the present study, treatment with AMF inhibited ovarian cell proliferation, increased P21 expression, decreased CDK1/2 expression, interrupted the balance of microtubule dynamics and arrested cells at the G2 phase. Furthermore, treatment with AMF increased the expression levels of phospho-Histone H2AX (γ-H2AX; a variant of histone 2A, that belongs to the histone 2A family member X) and the DNA repair protein RAD51 homolog 1 (Rad51), indicating the occurrence of DNA damage since γ-H2AX and Rad51 are both key markers of DNA damage. Consistent with previous findings, the results of the present study suggest that AMF is a potential therapeutic agent for the treatment of ovarian cancer. In addition, the effects of AMF on cell cycle arrest and DNA damage induction may be the molecular mechanisms by which AMF might exert its potential therapeutic benefits in ovarian cancer.

Amentoflavone: A Bifunctional Metal Chelator that Controls the Formation of Neurotoxic Soluble Aβ42 Oligomers

Alzheimer's disease (AD) is the most common neurodegenerative disorder, yet the cause and progression of this disorder are not completely understood. While the main hallmark of AD is the deposition of amyloid plaques consisting of the β-amyloid (Aβ) peptide, transition metal ions are also known to play a significant role in disease pathology by expediting the formation of neurotoxic soluble β-amyloid (Aβ) oligomers, reactive oxygen species (ROS), and oxidative stress. Thus, bifunctional metal chelators that can control these deleterious properties are highly desirable. Herein, we show that amentoflavone (AMF), a natural biflavonoid compound, exhibits good metal-chelating properties, especially for chelating Cu2+ with very high affinity (pCu7.4 = 10.44). In addition, AMF binds to Aβ fibrils with a high affinity (Ki = 287 ± 20 nM), as revealed by a competition thioflavin T (ThT) assay, and specifically labels the amyloid plaques ex vivo in the brain sections of transgenic AD mice, as confirmed via immunostaining with an Aβ antibody. The effect of AMF on Aβ42 aggregation and disaggregation of Aβ42 fibrils was also investigated and revealed that AMF can control the formation of neurotoxic soluble Aβ42 oligomers, both in the absence and presence of metal ions, as confirmed via cell toxicity studies. Furthermore, an ascorbate consumption assay shows that AMF exhibits potent antioxidant properties and can chelate Cu2+ and significantly diminish the Cu2+-ascorbate redox cycling and reactive oxygen species (ROS) formation. Overall, these studies strongly suggest that AMF acts as a bifunctional chelator that can interact with various Aβ aggregates and reduce their neurotoxicity and can also bind Cu2+ and mediate its deleterious redox properties. Thus AMF has the potential to be a lead compound for further therapeutic agent development for AD.

Amentoflavone, active compound of Selaginella tamariscina, inhibits in vitro and in vivo TGF-β-induced metastasis of human cancer cells

Epithelial mesenchymal transition (EMT) is a well-known and important step in metastasis and thus can be a key target in cancer treatment. Here, we tested the EMT inhibitory actions of Selaginella tamariscina and its active component, amentoflavone (AF). EMT was examined in vitro using wound-healing and invasion assays and by monitoring changes in the expression of the EMT-related proteins, E-cadherin, Snail, and Twist. Metastasis was examined in vivo using SCID mice injected with luciferase-labeled A549 cells. We confirmed that aqueous extracts of S. tamariscina (STE) and AF inhibited EMT in human cancer cell lines. We found that STE and AF at nontoxic concentrations exerted remarkable inhibitory effects on migration (wound healing assay) and invasion (Transwell assay) in tumor necrosis factor (TGF)-β–treated cancer cells. Western blotting and immunofluorescence imaging show that AF treatment also restored E-cadherin expression in these cells compared to cells treated with TGF-β only. Suppression of metastasis by AF was investigated by monitoring migration of tail-vein–injected, circulating A549-luc cells to the lungs in mice. After 3 wk, fewer nodules were observed in mice co-treated with AF compared with those treated with TGF-β only. Our findings indicate that STE and AF are promising EMT inhibitors and, ultimately, potentially potent antitumor agents.

Effects of structural differences on the antibacterial activity of biflavonoids from fruits of the Brazilian peppertree (Schinus terebinthifolius Raddi)

Flavonoids, synthesized by plants across all families and therefore found in a huge variety, possess a diverse range of pharmacological properties. Direct antibacterial and synergistic activities as well as the inhibition of several bacterial virulence factors are known. Besides the mode of action, it is important to understand the structure-activity relationship to identify key structural characteristics.This study aimed to identify biflavonoids with antibacterial activity from Schinus terebinthifolius Raddi fruits. The purified biflavonoids were characterized in terms of their antibacterial effects. We found that the activity of biflavonoids, including agathisflavone (AGF), amentoflavone (AMF), and tetrahydroamentoflavone (THAF), was dependent on their chemical configuration and degree of oxidation, with THAF showing the highest activity on planktonic cells. Additionally, biofilm formation and composition were strongly influenced by THAF.Even slight differences in the chemical structure have fundamental effects on the activity of isolated biflavonoids. This suggests a specific binding of these substances in bacteria and thus enables detailed investigations of the mode of action in the future.

RETRACTED: Amentoflavone suppresses cell proliferation and induces cell death through triggering autophagy-dependent ferroptosis in human glioma

AimsGlioma is the most common type of malignant tumor of the nervous system, and aggressiveness and recurrence are major obstacles for treatment. This study is designed to explore the effects of amentoflavone (AF) on glioma, and to investigate the underlying mechanism of the anti-cancer activities of AF. MethodsCell morphology was recorded under microscopy. Cell viability and cell death ratio were determined by CCK-8 assay and lactate dehydrogenase (LDH) release assay, respectively. Cell cycle progression was assessed by flow cytometry. The levels of iron, MDA (malondialdehyde), lipid ROS, and GSH (reduced glutathione) were assessed by ELISA kit. The cycle-related proteins, ferroptosis-related protein, autophagy-related protein, and the phosphorylation of AMPK, mTOR and p70S6K were analyzed by western blotting. The autophagic flux was observed by transfecting cells with mRFP-GFP-LC3 plasmids. The xenograft murine models were established to analyze the effects of amentoflavone in vivo. The immunohistochemistry assay was performed to analyze the expression of LC3B, Beclin1, ATG5, ATG7, and ferritin heavy chain (FTH). ResultsOur results showed that AF treatment led to reduction in cell viability and cell death. In addition, AF was found to block cell cycle progression in a dose-dependent manner in vitro. Following treatment with AF, the intracellular levels of iron, MDA, and lipid OS were increased, and the levels of GSH and the mitochondrial membrane potential were reduced. In addition, our results showed that AF promoted the autophagic by regulating autophagy-relevant proteins. Our results also showed that the autophagy-induction by AF was associated with regulation of AMPK/mTOR signaling. Mechanistically, the inhibition effects of AF on glioma cell were reversed by DFO, ferreostatin-1 as well as upregulation of FTH. Meanwhile, the FTH levels were increased by compound C and knockdown of ATG7. Moreover, both autophagy inhibitor Baf A1 and knockdown of ATG7 were able to compromising AF-induce ferroptosis and cell death. In vivo, the tumor growth was suppressed by AF in a dose-dependent manner. The level of MDA in the tumor tissue was increased while the level of GSH in tumor tissue was decreased by AF in a dose-dependent manner. Furthermore, the expression of LC3B, Beclin1, ATG5, ATG7 were increased, and the expression of FTH were decreased by AF in a dose-dependent manner in vivo. ConclusionThese results demonstrate that AF triggered ferroptosis in autophagy-dependent manner. Our results suggest that AF has the potential to be considered as a novel treatment agent in glioma.

Amentoflavone induces cell cycle arrest, apoptosis, and autophagy in BV-2 cells.

Previous studies have shown that amentoflavone (AF) elicits anti-inflammatory and neuroprotective effects. To further investigate the effects of AF on the microglia cell line BV-2, proteomic analysis was performed to screen potential key regulators. The top 5 canonical pathways associated with AF treatment were EIF2 signaling, regulation of eIF4 and p70s6k signaling, mTOR signaling, protein ubiquitination pathway and phagosome maturation. The top up-regulated genes were DOCK2, SEC23A, ME1, UGGT1 and STOM, while the most down-regulated molecules were IGF2R, ATP5O, DDX47, WBP11 and IKBIP. AF significantly decreased BV-2 cell proliferation. It induced cell cycle arrest at G2/M, increased CDK2, p27Kip1 and p53/p-p53, and decreased CDK1/CDC2 and cyclin B1. Cell apoptosis was induced, with increased levels of BAX, c-caspase-3 and c-caspase-9, and decreased levels of BCL-XL. Increased level of autophagosome induced by AF was observed, and increased Beclin-1 and decreased phosphorylation of PI3K and Erk1 were found as well. In conclusion, AF induces cell cycle arrest at G2/M, promotes apoptosis and autophagy in BV-2 cells, which may account for the anti-inflammatory effect of AF in epilepsy.

Preparation, evaluation and metabolites study in rats of novel amentoflavone-loaded TPGS/soluplus mixed nanomicelles

Amentoflavone (AMF) is a kind of biflavonoids existing in Ginkgo biloba leaves. It has many biological activities, such as antioxidant, anti-inflammatory, anti-bacterial, antiviral, hypoglycemic, anti-tumor and inducing apoptosis. However, its solubility and bioavailability are poor and there are a few studies on it in vivo. In this study, to improve its solubility and bioavailability, the nanomicelles were prepared with TPGS and soluplus as carriers for the first time. The particle size, Zeta potential, encapsulation efficiency, drug loading, stability, cytotoxicity, cellular uptake, and metabolites in rats were studied. Cytotoxicity, cellular uptake, and metabolites in rats of AMF-loaded TPGS/soluplus mixed micelles were compared with those of AMF. As a result, AMF-loaded TPGS/soluplus mixed micelles with a particle size of 67.33 ± 2.01 nm and Zeta potential of −0.84133 ± 0.041405 mV were successfully prepared. The encapsulation efficiency and drug loading of the mixed nanomicelles were 99.18 ± 0.76% and 2.47 ± 0.01%, respectively. The physical and chemical properties of the mixed micelles were stable within 60 d, and the cytotoxicity of the mixed micelles was much greater than that of AMF monomers. Thirty-four kinds of metabolites of AMF were identified in rats. The metabolites were mainly distributed in rat feces. No metabolites were detected in bile and plasma. 14 kinds of metabolites of the mixed micelles in rats were detected, including 11 in feces, 6 in urine, and 3 in plasma, which indicated that the bioavailability of AMF has been improved. And the toxicity to cancer cells was enhanced, which laid a foundation for the development of new drugs.

Amentoflavone ameliorates cold stress-induced inflammation in lung by suppression of C3/BCR/NF-\u03baB pathways

BackgroundCold stress, which may lead to local and systemic injury, is reported to be related to the immune system, especially the complement system. At present, the lack of effective treatment is a critical issue. Amentoflavone (AF), which can inhibit cold stress-induced inflammation in lung by multiple mechanisms, is the main therapeutic ingredient in plants of the genus Selaginella.ResultsIn the current study, we found that cold could induce lung inflammation related to the complement system and its downstream pathways. AF treatment significantly inhibited lung inflammation from cold exposure. We presented evidence that AF can bind to complement component 3 (C3) to regulate inflammation-related pathways involving Lck/Yes novel tyrosine kinase (Lyn), protein kinase B (Akt), nuclear factor-κB (NF-κB) and immune factors. Moreover, 30 mg/kg of AF caused significantly greater improvement than 15 mg/kg in reducing the level of C3 in lung tissue.ConclusionsAF can protect lung tissue from cold exposure. The protective effect may be achieved by inhibition of C3 and negative regulation of the B cell receptor (BCR)/NF-κB signaling pathways and high mobility group box 1 (HMGB1), which ultimately ameliorates the inflammatory response.

Amentoflavone suppresses amyloid β1–42 neurotoxicity in Alzheimer's disease through the inhibition of pyroptosis

BackgroundThe accumulation of Amyloid β (Aβ) plays key roles in Alzheimer's disease (AD) by inducing intracellular reactive oxygen species (ROS) and neuronal cell death. In this study, we aimed to identify the neuroprotective mechanisms of amentoflavone (AF) in Aβ-induce neuronal cell injury. Materials and methodsThe animal model was established by injecting Aβ1-42 into the bilateral hippocampus. The effect of AF on Aβ1-42-induced neurological dysfunction was examined using the Y-maze and radical maze tests. The hippocampal neuron viability was examined using Nissl staining and TUNEL assay. On the other hand, in vitro studies were conducted using SH-SY5Y cells. The expression level of marker proteins was measured using western blot. The activity of caspase-1 and the levels of pro-inflammatory cytokines were determined using ELISA assay. AMPKα knock down was carried out by transfecting SH-SY5Y cells with siRNA against AMPK transcript. ResultsNeurological tests showed that AF significantly attenuated Aβ1-42-induced neurological dysfunction. AF suppressed Aβ1-42-induced pyroptosis in the hippocampal region of the rat model, which was associated with the modulation of AMPK/GSK3β signaling. Similar results were obtained in vitro in SH-SY5Y cells exposed to Aβ1-42, showing that the neuroprotective activity of AF is mediated by suppressing pyroptosis through AMPK/GSK3β signaling. ConclusionAF inhibits Aβ1-42-induced neurotoxicity in animal and cellular models through AMPK/GSK3β-mediated pyroptosis suppression. Our results highlight AF as a clinical compound for the prevention and treatment of AD.

Amentoflavone Promotes Apoptosis in Non-Small-Cell Lung Cancer by Modulating Cancerous Inhibitor of PP2A.

Non-small-cell lung cancer (NSCLC) is one of the most common human malignancies. Amentoflavone (AF) is one of bioflavonoid compounds isolated from Selaginella tamariscina Spring. This study was designed to examine the effect of AF on NSCLC. Our results indicated that AF decreased cell viability of both H1299 and H358 cells. Colony formation assay also showed that AF was able to suppress the anchorage-independent growth of NSCLC cells. AF also triggered cell cycle arrest by downregulating cyclin D1, CDK4, and CDK6. The pro-apoptotic activity of AF was confirmed by Hoechst staining and flow cytometry. The effect of AF on activation of caspase-3, upregulation of Bax, and downregulation of Bcl-2 was examined by western blot. The anti-growth and pro-apoptotic activities of AF were further validated in xenograft murine model. iTRAQ assay showed that cancerous inhibitor of PP2A (CIP2A) expression was markedly downregulated by AF treatment in H1299 cells. In addition, qRT-PCR and western blot also showed that AF was able to dose-dependently inhibit CIP2A expression. Meanwhile, the activity of protein phosphatase 2A (PP2A) was enhanced by AF treatment. The mRNA and protein expression of CIP2A as well as PP2A activity in xenograft tumor tissue were examined, which indicated that the in vivo anticancer activity of AF was associated with downregulation of CIP2A and reactivation of PP2A. Moreover, our results showed that the anti-growth and pro-apoptotic activities of AF were augmented by CIP2A knockdown and attenuated by ectopic CIP2A expression. Our results indicated that AF exhibited anticancer activity in NSCLC by targeting CIP2A. Anat Rec, 302:2201-2210, 2019. © 2019 American Association for Anatomy.

Amentoflavone protects the hematopoietic system of mice against γ-irradiation.

Some flavonoids have been shown to exhibit good antioxidant activity and protect mice from damage induced by radiation. Amentoflavone (AMF), a biflavonoid derived from the traditional herb-Selaginella tamariscina, has been reported to have antioxidant properties. The protective effects and mechanism of action of AMF against radiation injury remain unknown. In this study, male C57BL/6 mice were subjected to total-body 60Co γ-irradiation at 7.5 or 3.0Gy. The survival rate and mean survival time were evaluated to determine the radioprotective effect of AMF. Number of peripheral blood cells, frequency of colony forming unit-granulocytes, monocytes and micronuclei were measured to assess the protective effects of AMF on the hematopoietic system. Levels of superoxide dismutase and glutathione, and pathological changes in the bone marrow were determined. Additionally, next-generation sequencing technology was used to explore potential targets of AMF. We observed that AMF markedly extends average survival time, reduces injury to the hematopoietic system and promotes its recovery. Furthermore, treatment with AMF significantly attenuated radiation-induced oxidative stress. In addition, AMF had a significant effect on gene tumor necrosis factor alpha-induced protein 2. Together, the results of this study suggest that AMF is a potential protective agent against radiation injury.

Ionic Liquid-Microwave-Based Extraction of Biflavonoids from Selaginella sinensis

Selaginella sinensis (Desv.) Spring has been used for many years as traditional Chinese medicine (TCM) for many years. Recently, ionic liquids (ILs) have attracted great attentions in extraction and separation technology of TCM as a new green solvent. In this paper, microwave assisted extraction-IL (MAE-IL) that extracted amentoflavone (AME) and hinokiflavone (HIN) from Selaginella sinensis was reported for the first time. The contents of two biflavonoids were simultaneously determined by a high performance liquid chromatography (HPLC) method. After different ionic liquids were compared, it was found [C6mim]BF4 had a high selectivity and efficiency. Moreover, the important extraction conditions, including solid-liquid ratio, IL concentration, extraction time, microwave power and radiation temperature, were also investigated and optimized by response surface methodology (RSM) using AME and HIN yields as index. The results showed that the extraction yields of AME and HIN from S. sinensis were 1.96 mg/g and 0.79 mg/g, respectively, under the optimal process parameters (0.55 mmol/L, 300 W, 40 min, 1:11 g/mL and 48 °C). Compared with the conventional extraction methods, MAE-IL could not only achieve higher yield in shorter time, but also could reduce the consumption of solvent. This effective, rapid and green MAE-IL method was suitable for the extraction of AME and HIN.

Antidiabetic Activity and Potential Mechanism of Amentoflavone in Diabetic Mice.

Aim: To investigate the anti-diabetic activity of amentoflavone (AME) in diabetic mice, and to explore the potential mechanisms. Methods: Diabetic mice induced by high fat diet and streptozotocin were administered with amentoflavone for 8 weeks. Biochemical indexes were tested to evaluate its anti-diabetic effect. Hepatic steatosis, the histopathology change of the pancreas was evaluated. The activity of glucose metabolic enzymes, the expression of Akt and pAkt, and the glucose transporter type 4 (GLUT4) immunoreactivity were detected. Results: AME decreased the level of glucose, total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and glucagon, and increased the levels of high density lipoprotein cholesterol (HDL-C) and insulin. Additionally, AME increased the activity of glucokinase (GCK), phosphofructokinase-1 (PFK-1), and pyruvate kinase (PK), and inhibited the activity of glycogen synthase kinase-3 (GSK-3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G-6-Pase). Mechanistically, AME increased superoxide dismutase (SOD), decreased malondialdehyde (MDA), activation of several key signaling molecules including pAkt (Ser473), and increased the translocation to the sedimenting membranes of GLUT4 in skeletal muscle tissue. Conclusions: AME exerted anti-diabetic effects by regulating glucose and lipid metabolism, perhaps via anti-oxidant effects and activating the PI3K/Akt pathway. Our study provided novel insight into the role and underlying mechanisms of AME in diabetes.

Identification of amentoflavone as a potent highly selective PARP-1 inhibitor and its potentiation on carboplatin in human non-small cell lung cancer

Nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1) is a key enzyme in the repair of DNA and is a promising target in the development of chemosensitizers. This study first investigated the inhibitory effects of amentoflavone (AMF) and its derivatives on PARP-1 and the potentiation of AMF on carboplatin (CBP) in non-small cell lung cancer (NSCLC). This study aims to evaluate the inhibitory effect of AMF against PARP-1 and its potentiation on CBP in lung cancer both in vitro and in vivo. The inhibitory effect of AMF on PARP-1 was investigated using molecular docking and cell-free model of PARP-1 assay. Its potentiation on CBP in lung cancer was also evaluated. Fluorescence resonance energy transfer assay was used to detect the inhibitory effects of AMF and its analogues on PARP-1. Molecular docking was employed to predict the binding mode of AMF and PARP-1. MTT assay, isobologram analysis, Hoechst staining, and Annexin V-PI double staining were used to confirm the potentiation of AMF on CBP in vitro. siRNA (PARP-1)-A549 cells were used to reveal the action target of AMF. Western blot analysis, immunohistochemistry, and Tunnel assay were employed to evaluate the potentiation of AMF on CBP in A549 xenograft mice. AMF and its analogues exerted excellent inhibitory effects on PARP-1 with IC50 values ranging from 0.198 μM to 0.409 μM. Docking experiment showed that AMF can stably bind to PARP-1 with a comparable binding energy to olaparib. AMF can decrease the expression of PAR induced by H2O2in vitro. AMF synergistically increased the CBP anti-proliferative effect in A549. However, its potentiation nearly disappeared when the cells were transfected with siRNAs against PARP-1. Oral administration of AMF (100 mg/kg), combined with CBP, remarkably inhibited A549 tumor growth and ki67 expression, and increased apoptosis compared with CBP-alone group. All results suggest that AMF can be a potential PARP-1 inhibitor and a candidate adjuvant agent to boost the anticancer effect of CBP in NSCLC.

Amentoflavone induces apoptosis and suppresses glycolysis in glioma cells by targeting miR-124-3p

Malignant glioma is the most common type of brain tumor with poor clinical outcome and survival. Therefore, it is imperative to develop novel therapeutic agents for managing glioma. The aim of this study was to investigate the role of amentoflavone (AF), an active flavonoid component in Selaginella tamariscina Spring, in glioma cells and the underlying mechanism of its action. Our results showed that miR-124-3p expression was significantly down-regulated in glioma tissues relative to normal brain tissues. AF decreased cell viability and triggered apoptosis in both glioma cell lines in a dose-dependent manner. AF induced apoptosis and inhibited glycolysis in the glioma cells by upregulating miR-124-3p. Furthermore, AF upregulated miR-124-3p by repressing DNMT1 through Sp1, which in turn was caused by the activation of ROS/AMPK signaling pathway by AF. In conclusion, AF could induce apoptosis and inhibited glycolysis in glioma cells via miR-124-3p. Our findings provide preliminary experimental data that support further investigation on the therapeutic efficacy of AF in glioma.

Abstract 5428: Effect of amentoflavone on Nrf2 signaling in HaCaT cells

Abstract Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key mediator of cell response to oxidative stress and xenobiotics. Multiple lines of evidence suggest that Nrf2 activation protects against many diseases, aging, inflammation, and cancer. To date, Nrf2 activators from natural products have been widely studied. In this study we investigated the potential effect of Amentoflavone (AFN), a biflavonoid in many natural plants, on Nrf2 signaling in human keratinocytes cells (HaCaT). As results, AFN significantly increased ARE-luciferase activity and nuclear accumulation of Nrf2. Furthermore, a Nrf2 target protein, NQO-1, was significantly increased by AFN in dose- and time-dependent manner. To verify the Nrf2 signaling mechanism by AFN, firstly we measured reactive oxygen species (ROS) using DCFDA because mild intracellular ROS could stimulate Nrf2 activation. Surprisingly AFN triggered the production of ROS in 1 h and AFN-induced Nrf2 was inhibited by N-acetyl cysteine (NAC). Thus, we studied the ROS-related signaling on Nrf2 by measuring the activation of Akt and mitogen-activated protein (MAP) kinase family such as extracellular signal-regulated kinase (ERK) and p38. As results, pharmacology inhibitor of PI3K/Akt (LY294002) or p38 (SB 203580), but not ERK (U0126), abrogated AFN-activated Nrf2. Furthermore, silencing p38 using p38siRNA or inhibition using its pharmacology inhibitor decreased phosphorylation of Akt and attenuated Nrf2 activation. We also confirmed that AFN stabilized Nrf2 by inhibiting the ubiquitination. Taken together, AFN activates Nrf2 through ROS-mediated phosphorylation of p38 and followed by phosphorylation of Akt. Citation Format: Lilik Duwi Wahyudi, Jiwon Jeong, Heejung Yang, Jung-Hwan Kim. Effect of amentoflavone on Nrf2 signaling in HaCaT cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5428.

Amentoflavone-induced oxidative stress activates NF-E2-related factor 2 via the p38 MAP kinase-AKT pathway in human keratinocytes.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that responds to oxidative stress and xenobiotics. Multiple lines of evidence suggest that Nrf2 activation protects against aging, inflammation, and many diseases, including cancer. Nrf2 activators derived from natural sources have been widely studied. In this study, we investigated the effect of amentoflavone (AFN), a biflavonoid found in many plants, on Nrf2 signaling in human keratinocytes (HaCaT cells). AFN significantly increased ARE luciferase activity by Nrf2 accumulation in the nucleus. Subsequently, the levels of a Nrf2 target protein, NQO-1, were significantly increased by AFN in a dose- and time-dependent manner. To verify the mechanism of AFN-induced activation of Nrf2 signaling, we measured generation of reactive oxygen species (ROS). Interestingly AFN triggered mild ROS production. Additionally, AFN-induced Nrf2 activation was inhibited by N-acetyl cysteine. Therefore, we studied the effect of ROS-related signaling on Nrf2 by measuring the activation of AKT and members of the mitogen-activated protein kinase family, such as extracellular signal-regulated kinase (ERK1/2) and p38. The results showed that the pharmacological inhibitor of PI3K/AKT (LY294002) or p38 (SB 203580), but not ERK1/2 (U0126), abrogated AFN-induced Nrf2 activation. Subsequently, we found that silencing or inhibition of p38 resulted in decrease of AKT phosphorylation as well as inhibition of Nrf2 accumulation. Furthermore, we found that AFN stabilized Nrf2 by inhibiting its ubiquitination. Taken together, our results suggest that AFN contributes to Nrf2 activation through ROS-mediated activation of the p38-AKT pathway in HaCaT cells.

Amentoflavone is a potent broad-spectrum inhibitor of human UDP-glucuronosyltransferases

Amentoflavone (AMF), an abundant natural biflavonoid found in many medicinal plants, displays various beneficial effects including anti-inflammatory, anti-oxidative and anti-cancer. Despite the extensive studies on pharmacological activities, the toxicity or undesirable effects of AMF are rarely reported. In this study, the inhibitory effects of AMF on human UDP-glucuronosyltransferases (UGTs) were carefully investigated. AMF displayed strong inhibition towards most of human UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4 and 2B17, with the IC50 values ranging from 0.12 μM to 16.81 μM. Inhibition constants (Ki) of AMF against various human UGTs varied from 0.29 μM to 11.51 μM. Further investigation demonstrated that AMF was a noncompetitive inhibitor of UGT1A1 mediated NCHN-O-glucuronidation but functioned as a competitive inhibitor of UGT1A1 mediated 4-MU-O-glucuronidation. In addition, AMF was a competitive inhibitor of UGT1A4 mediated TFP-N-glucuronidation in both UGT1A4 and human liver microsomes, while functioned as a competitive inhibitor of UGT1A9 mediated propofol or 4-MU-O-glucuronidation. These findings demonstrated that AMF was a strong and broad-spectrum natural inhibitor of most human UGTs, which might bring potential risks of herb-drug interactions (HDIs) via UGT inhibition. Additionally, this study provided novel insights into the underlying mechanism of AMF-associated toxicity from the perspective of UGT inhibition.

Amentoflavone Ameliorates Aβ1-42-Induced Memory Deficits and Oxidative Stress in Cellular and Rat Model.

Alzheimer's disease (AD), a progressive neurodegenerative disease of the central nervous system, is the most common cause of senile dementia. This study aimed to investigate whether amentoflavone (AF), a biflavonoid compound, could exert neuroprotective activities against AD. The AD model was established by the intracranial injection of amyloid-beta (Aβ) in rat models. The effect of AF on cognitive function was examined using the Morris water maze test. Cell survival and apoptosis in the hippocampal region in an animal model were detected using Nissl staining and a terminal deoxynucleotidyl transferased UTP nick-end labeling assay, respectively. The levels of oxidant enzymes were determined by enzyme-linked immunosorbent assay. Signaling molecule expressions were examined by western blotting. Our results showed that AF significantly attenuated Aβ-induced deficits in neurological functions as well as neuronal cell death and apoptosis in the hippocampal region. Moreover, our findings revealed that AF increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and translocation and activated AMP-activated protein kinase (AMPK) signaling. In a cellular model of AD established by exposing PC12 cells to Aβ, our results provided further evidence that the neuroprotective activities of AF were mediated by modulating Nrf2 through AMPK/glycogen synthase kinase 3 beta signaling. AF exerts a protective effect against Aβ1-42-induced neurotoxcicity by inducing Nrf2 antioxidant pathways via AMPK signaling activation, which provided experimental evidence that AF might provide a clinical benefit to patients with AD.