Hybrid pharmacophore design and synthesis of donepezil-inspired aurone derivative salts as multifunctional acetylcholinesterase inhibitors.

Flavonoids, a ubiquitous group of plant polyphenols, are well-known for their beneficial effects on human health. Their phenylchromane skeletons have structural similarities to donepezil [the US FDA-approved drug used to treat Alzheimer's disease (AD)]. The objective of this study was to design and synthesize valuable agents derived from flavonoids for relieving the symptoms of AD. A variety of flavonoid derivative salts incorporating benzylpyridinium units were synthesized and several of them remarkedly inhibited acetylcholinesterase (AChE) activity in vitro. Additionally, aurone derivative salts protected against cell death resulting from t-BHP exposure in rat pheochromocytoma PC12 cells and slightly promoted neurite outgrowth. Furthermore, they potently suppressed the aggregation of amyloid-ß (Aß1-42). Our findings highlight the effectiveness of donepezil-inspired aurone derivative salts as multipotent candidates for AD.

Promotion of ABCG2 gene expression by neolignans from Piper longum L.

We focused on Piper longum L., a herbal drug produced in Myanmar, which has a renoprotective effect. Thus, we attempted to isolate and identify compounds that enhance the expression of the ABCG2 gene from the aerial parts of the plant except for the fruit. Among the various P. longum extracts, we isolated and identified the components. Using Caco-2 cells, the hABCG2 mRNA expression-enhancing effects of the isolated compounds were compared with the positive reference compound (3-methylcholanthrene [3MC]) using real-time polymerase chain reaction. Six compounds were isolated and identified from the methanol extract of P. longum. Among the isolated compounds, licarin A and neopomatene had lower toxicity and higher hABCG2 mRNA expression-enhancing effects in Caco-2 cells. Suppression of hAhR expression by siRNA reduced the activity of licarin A and neopomatene, as well as the hAhR agonist 3MC, suggesting that these 2 compounds may act as hAhR agonists to promote hABCG2 expression.

Synthesis of raloxifene-like quinoxaline derivatives by intramolecular electrophilic cyclization with disulfides.

The intramolecular electrophilic cyclization of alkynes with disulfides to form thieno[2,3-b]quinoxaline structures and to introduce thioether substituents afforded quinoxaline derivatives (7a-7d, 8a-8d). Among obtained eight derivatives, the raloxifene analogues (7c, 8b) showed specifically high cytotoxicity against breast cancer cells (SK-BR-3), and raloxifene analogues (8a) showed the highest cytotoxicity against human leukemia cells (HL-60). None of the raloxifene analogues (7a-7d, 8a-8d) showed cytotoxicity against human lung fibroblasts (WI-38), which are normal cells.

Amino Acid Conjugates of ent-Beyerane-type Diterpenoids and Oleanane-type Triterpenoids: Synthesis and Evaluation of Cytotoxic Activities.

The aim of this study was to increase the cytotoxic activities of terpenoids via amino acid conjugation. Thus, 21 new ester derivatives (5-15, 19-27, and 29) were prepared by conjugation of the hydroxy groups in ent-beyerane-type diterpenoids (4) and oleanane-type triterpenoids (18), and their cytotoxic activity against three human cancer cell lines (leukemia, lung, and stomach) were evaluated. The prepared compounds showed cytotoxic effects; in particular, all amino acid conjugates of ent-beyerane-type diterpenoids (5-13) exhibited potent cytotoxic activity (IC50 1.0-3.7 µM for HL60, 1.7-8.2 µM for A549, and 2.5-11.7 µM for MKN45). In addition, no differences were observed in the cytotoxic activities of l- and d-type amino acid conjugates.

Molecular Evolution of the Pseudomonas aeruginosa DNA Gyrase gyrA Gene.

DNA gyrase plays important roles in genome replication in various bacteria, including Pseudomonasaeruginosa. The gyrA gene encodes the gyrase subunit A protein (GyrA). Mutations in GyrA are associated with resistance to quinolone-based antibiotics. We performed a detailed molecular evolutionary analyses of the gyrA gene and associated resistance to the quinolone drug, ciprofloxacin, using bioinformatics techniques. We produced an evolutionary phylogenetic tree using the Bayesian Markov Chain Monte Carlo (MCMC) method. This tree indicated that a common ancestor of the gene was present over 760 years ago, and the offspring formed multiple clusters. Quinolone drug-resistance-associated amino-acid substitutions in GyrA, including T83I and D87N, emerged after the drug was used clinically. These substitutions appeared to be positive selection sites. The molecular affinity between ciprofloxacin and the GyrA protein containing T83I and/or D87N decreased significantly compared to that between the drug and GyrA protein, with no substitutions. The rate of evolution of the gene before quinolone drugs were first used in the clinic, in 1962, was significantly lower than that after the drug was used. These results suggest that the gyrA gene evolved to permit the bacterium to overcome quinolone treatment.

Rapid removal of detergent in glycolipids using ionic liquids.

Detergent removal in glycolipid after sample preparation, such as enzymatic reaction or isolation of detergent-resistant membrane microdomain, is indispensable for further structural characterization. We previously established the rapid and effective method of detergent removal in glycolipid samples from glass test tube using 1,2-dichloroethane (DCE) washing. However, the use of DCE has several drawbacks, such as environmental risks, harmful effects (potentially carcinogenic), and high vaporability and flammability. To solve the issue, we used ionic liquids to remove detergents from glycolipid samples, and found 1-butyl-3-methylimidazolium iodide was a suitable alternative for DCE.

Effects of Dapagliflozin on Adipose and Liver Fatty Acid Composition and mRNA Expression Involved in Lipid Metabolism in High-Fat-Fed Rats.

BACKGROUND: SGLT2 inhibitor enhances not only glucose excretion but also fatty acid utilization. Those facts suggest that SGLT2 inhibitor affects fat accumulation and lipid storage. OBJECTIVE: In the present study, we evaluated the effects of dapagliflozin on fatty acid composition and gene expression involved in fatty acid metabolism in rat adipose and liver tissues. METHODS: We administered 1 mg/kg/day dapagliflozin for 7 weeks to male high-fat-fed rats (DAPA group), and then weights and 22 fatty acid contents in the epididymal (EPI), mesenteric (MES), retroperitoneal (RET), and subcutaneous (SUB) adipose tissues, and the liver were compared with the vehicle-administered control group. RESULTS: In the EPI, RET, and SUB in the DAPA group, contents of several fatty acids were lower (P<0.05) than those in the control group, while no significant difference was detected in tissue weight. In the MES, tissue weight and a wide variety of fatty acid contents, including saturated, monounsaturated, and polyunsaturated fatty acids, were lower (P<0.05). As for the liver tissue, no significant difference was observed in fatty acid contents between the groups. mRNA expression of Srebp1c in EPI was significantly higher (P<0.05) in the DAPA group than in the control group, while Scd1 expression in the liver was lower (P<0.01). CONCLUSION: These results suggest that dapagliflozin might suppress lipid accumulation especially in the MES, and could reduce contents of fatty acids not in the liver but in adipose tissues in high-fat-fed rats. In addition, dapagliflozin could influence mRNA expression involved in lipogenesis in the EPI and liver.

Effects of dapagliflozin on peripheral sympathetic nerve activity in standard chow- and high-fat-fed rats after a glucose load.

To clarify the effects of long-term administration of SGLT2 inhibitor, a hypoglycemic agent, on basal sympathetic nerve activity (SNA) and on SNA under development of insulin resistance, we measured peripheral SNA in response to a glucose load in standard chow- (SCF) and high-fat-fed (HFF) rats treated with or without dapagliflozin for 7 weeks. We conducted an intravenous glucose administration (IVGA), and evaluated SNA microneurographically recorded in the unilateral sciatic nerve. Dapagliflozin did not affect the steady state action potential (AP) rate just before the IVGA (baseline) in both the SCF and HFF rats. After the IVGA, in the SCF rats, the AP rate in dapagliflozin-treated group transiently decreased within 20 min after the IVGA, and was significantly lower (P < 0.05) than non-treated group for 60 min. In the HFF rats, no significant difference was seen in the AP rate between dapagliflozin-treated and non-treated groups. The rate in the dapagliflozin-treated group after the IVGA was significantly lower (P < 0.05) than the baseline whereas such difference was not found in the non-treated group. In conclusion, dapagliflozin attenuate SNA in response to glucose load, and that the SNA response is different between standard chow-fed- and high-fat-fed rats.

Effect of Compounds from Moringa oleifera Lam. on in Vitro Non-Alcoholic Fatty Liver Disease (NAFLD) Model System.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease in the world, with a prevalence of 25 % in many countries. To date, no drug has been approved to treat NAFLD, therefore, the use of phytochemicals to prevent this disease is meaningful. In this study, we focused on the effects of Moringa oleifera Lam. on diabetes, attempted to isolate compounds that regulate NAFLD. Compounds 1 and 2 were isolated from the ethyl acetate fraction of M. oleifera. Spectral data revealed that they were 1-hydroxy-3-phenylpropan-2-yl benzoate (1) and benzyl benzylcarbamate (2), respectively. The three-dimensional structure of compound 1 was determined by single crystal X-ray structural analysis. Neither compound was toxic to HepG2 cells, and compound 1 was found to have a concentration-dependent inhibitory effect on intracellular lipid accumulation induced by stimulation of linoleic acid (LA). As a result of measuring the effects of compound 1 on the intracellular lipid production-related protein, it was found that compound 1 enhanced protein expression that promotes lipolysis. On the other hand, since the action of compound 1 was similar to that of PPARα agonists, it is deduced that compound 1 enhanced the activity of PPARα and further enhanced the expression of lipolytic proteins, which is related to the suppression of intracellular lipid accumulation. Furthermore, as the result of docking simulation, compound 1 had a higher binding affinity to the ligand binding site of PPARα than fenofibrate, which is a PPARα agonist, and thus compound 1 was considered to be promising as an agonist of PPARα.

Detailed Molecular Interactions of Favipiravir with SARS-CoV-2, SARS-CoV, MERS-CoV, and Influenza Virus Polymerases In Silico.

Favipiravir was initially developed as an antiviral drug against influenza and is currently used in clinical trials against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (COVID-19). This agent is presumably involved in RNA chain termination during influenza virus replication, although the molecular interactions underlying its potential impact on the coronaviruses including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) remain unclear. We performed in silico studies to elucidate detailed molecular interactions between favipiravir and the SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza virus RNA-dependent RNA polymerases (RdRp). As a result, no interactions between favipiravir ribofuranosyl-5'-triphosphate (F-RTP), the active form of favipiravir, and the active sites of RdRps (PB1 proteins) from influenza A (H1N1)pdm09 virus were found, yet the agent bound to the tunnel of the replication genome of PB1 protein leading to the inhibition of replicated RNA passage. In contrast, F-RTP bound to the active sites of coronavirus RdRp in the presence of the agent and RdRp. Further, the agent bound to the replicated RNA terminus in the presence of agent, magnesium ions, nucleotide triphosphate, and RdRp proteins. These results suggest that favipiravir exhibits distinct mechanisms of action against influenza virus and various coronaviruses.

Synthesis of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole derivatives as potent antiproliferative agents via a hybrid pharmacophore approach.

Imiquimod (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine) is efficacious in topical therapy for certain types of skin cancers. Structurally similar EAPB0203 (N-methyl-1-(2-phenethyl)imidazo[1,2-a]quinoxalin-4-amine) has been shown higher in vitro potency than imiquimod. Besides, triazole, oxadiazole, and thiadiazole rings are privileged building blocks in drug design. A series of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole and [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-thiadiazole derivatives were therefore synthesized by incorporation of these rings into the structure of EAPB0203 and assessed their antiproliferative effects against various cancer cell lines. The 1,3,4-oxadiazole derivatives demonstrated the superior effectiveness compared to imiquimod and EAPB0203. Our findings highlight the excellent potential of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole derivatives as anticancer agents.

Design and synthesis of quinoxaline-1,3,4-oxadiazole hybrid derivatives as potent inhibitors of the anti-apoptotic Bcl-2 protein.

Quinoxaline is one of the privileged heterocyclic fragments for drug molecules. Quinoxaline anticancer drug candidates XK469 and CQS exhibit antiproliferative and proapoptotic properties against various cancers. Based on their chemical structures, we therefore synthesized a series of quinoxaline-1,3,4-oxadiazole hybrids and assessed their anticancer potential on human leukemia HL-60 cells. Although these hybrids exerted significant inhibition of HL-60 cell proliferation, they showed high cytotoxicity on human normal cells (WI-38). Utilizing information from molecular modelling of the hybrids to the anti-apoptotic Bcl-2 protein, we added substructures including phenyl, piperazine, piperidine, and morpholine rings to their frameworks. The designed quinoxaline-1,3,4-oxadiazole hybrid derivatives successfully induced apoptotic response on HL-60 cells with low toxicity on WI-38 cells. Furthermore, RT-PCR analysis demonstrated that these derivatives predominantly inhibit Bcl-2 expression. Our findings highlight the great potential for the development of synthetic quinoxaline-1,3,4-oxadiazole hybrid derivatives as proapoptotic anticancer agents.

Tokoronin Contained in Dioscorea tokoro Makino ex Miyabe Suppressed α-MSH-Induced Melanogenesis in B16 Cells via Suppression of Classical MAPK Pathway Activation.

In this study, melanogenesis inhibition in B16 cells by eight compounds, namely, tokorogenin, tokoronin, yononin, gracillin, proto-yonogenin, proto-tokoronin, proto-yononin, and proto-gracillin, isolated from Dioscorea tokoro Makino ex Miyabe were evaluated. The results of the cytotoxicity and α-MSH-induced melanogenesis inhibition effects of the eight compounds revealed that tokoronin was the most effective in terms of low-cytotoxicity and melanogenesis inhibition. Tokoronin downregulated α-MSH-induced melanogenesis via suppression of the expression of the three types of melanogenesis-related enzymes [tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2] by the inhibition of phospho-microphthalmia-associated transcription factor (p-MITF) and cAMP response element binding protein (CREB) levels. p-MITF and CREB are regulated by various kinases [Akt, mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK)]. As the results of measurement of the combined effects of tokoronin with inhibitors or promoters of these kinases, no change in the biological activity of tokoronin by Akt inhibitor (wortmannin) or p38 MAPK inhibitor (SB202190) was observed, however, the effect of tokoronin was reduced by the MEK/ERK inhibitor (U0126) and promoted by the MEK/ERK activator (FGF2). Therefore, it was deduced that tokoronin first inactivated ERK; then, it suppressed p-MITF and CREB levels; and finally, α-MSH-induced melanogenesis was suppressed.

The Effect of Methanolic Valeriana officinalis Root Extract on Adipocyte Differentiation and Adiponectin Production in 3T3-L1 Adipocytes.

Adipose tissue is an endocrine organ and its endocrine function is closely associated with type 2 diabetes mellitus. Valeriana officinalis (Valerian) exerts some physiological effects; however, its influence on adipocytes remains unclear. We investigated the effect of methanolic Valerian root extract (Vale) on 3T3-L1 adipocytes. Vale (1, 10, and 100 µg/mL) dose-dependently promoted adipocyte differentiation with increasing lipid accumulation. In addition, Vale significantly increased the mRNA levels in genes associated with adipocyte differentiation, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α , and adipocyte protein 2, in dose-dependent manner. Vale also significantly enhanced mRNA and protein levels in adiponectin. A PPARγ antagonist assay and a PPARγ binding assay revealed that Vale-induced increased adipocyte differentiation and adiponectin production were partly associated with direct binding to PPARγ. Valerenic acid, a characteristic component in Valerian, also demonstrated the ability to induce adipocyte differentiation and adiponectin secretion, suggesting that it is one of the functional components in Vale.

(-)-O-Methylcubebin from Vitex trifolia Enhanced Adipogenesis in 3T3-L1 Cells via the Inhibition of ERK1/2 and p38MAPK Phosphorylation.

In this study, for the purpose of elucidation for antidiabetic components, we isolated and identified compounds that could become lead compounds for the development of antidiabetic agents from the herbal medicine Vitex trifolia, which is used for liver protection in Myanmar. Three kinds of lignan, (-)-O-methylcubebin (MC), (-)-hinokinin, and (-)-cubebin, were isolated from the ethyl acetate extract of the leaves of V. trifolia, using various chromatography. Among the three isolated compounds, MC showed the strongest effects to increase intracellular lipid accumulation in 3T3-L1 cells. From the results of the elucidation of the MC's effects on the adipogenesis of 3T3-L1 cells, the downsizing of adipocytes and the promotion of the expression of adipogenesis-related proteins, as well as adiponectin, were observed. On the other hand, since the activity of MC was inhibited by antagonists of PPARγ and improved by inhibitors of the classical mitogen-activated protein kinase (MAPK) pathway and p38MAPK pathway, MC was considered to be an agonist of PPARγ, and furthermore promoted adipogenesis via the inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38MAPK phosphorylation. Although MC showed similar effects to those of rosiglitazone (RO) used as a positive control, RO promoted the migration of GLUT4 from the cytoplasm to the cell membrane, whereas MC did not show such an effect. From the abovementioned results, it was considered that MC could be a lead compound for the development of antidiabetic drugs that does not show weight gain, which is a side effect of RO.

Comparative analysis of stilbene and benzofuran neolignan derivatives as acetylcholinesterase inhibitors with neuroprotective and anti-inflammatory activities.

Stilbenes and benzofuran neolignans are important groups of plant phenolics therefore they play a significant role in plants and human health. The objective of this study was to investigate the structure-activity relationships of naturally occurring stilbene and benzofuran neolignan derivatives as acetylcholinesterase inhibitors. A series of these compounds were prepared and assessed for their inhibition on acetylcholinesterase activity. δ-Viniferin, pterostilbene trans-dehydrodimer, pallidol, grossamide, and boehmenan exerted acetylcholinesterase inhibitory potential. The several oligomeric compounds protected against cell damage resulting from t-BHP exposure and inhibited lipopolysaccharide/interferon-gamma (LPS/IFNγ)-induced NO production in vitro. Our findings highlight the great potential of pterostilbene trans-dehydrodimer, pallidol, and boehmenan as multifunctional nutraceuticals for management of neurodegenerative diseases.

Antidiabetic-Like Effects of Naringenin-7-O-glucoside from Edible Chrysanthemum 'Kotobuki' and Naringenin by Activation of the PI3K/Akt Pathway and PPARγ.

Obesity is directly associated with cancer, cardiovascular injury, hypertension, and type 2 diabetes. To date, Yamamoto identified that hot water extracts of edible Chrysanthemum (EC) induced cell size reduction, up-regulation of adiponectin expression, and glucose absorption inhibition in 3T3-L1 cells during adipocyte differentiation. Furthermore, EC showed antidiabetic effects such as improvement in insulin resistance and the down-regulation of the blood glucose level and liver lipid content in type 2 diabetes model mice. In this study, we attempted to identify the antidiabetic components in EC. The methanol fraction from EC that showed relatively strong biological activity was purified by chromatography to obtain acacetin-7-O-glucoside, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, and naringenin-7-O-glucoside. Among the isolated compounds and their aglycones, naringenin (NA) and naringenin-7-O-glucoside (NAG) up-regulated the intracellular accumulation of lipid and adiponectin-secretion and down-regulated the diameter of 3T3-L1 cells during adipocyte differentiation. Because the PPARγ antagonist BADGE and PI3K/Akt inhibitors wortmannin and LY29004 inhibited the intracellular lipid accumulation by NA and NAG associated with adipogenesis, it was considered that NA and NAG showed the above-mentioned activities via the activation of PPARγ as well as phosphorylation of the PI3K/Akt pathway.

Mansonone E from Mansonia gagei Inhibited α-MSH-Induced Melanogenesis in B16 Cells by Inhibiting CREB Expression and Phosphorylation in the PI3K/Akt Pathway.

Many natural products that inhibit melanogenesis, freckles, and hyperpigmentation have been selectively used in cosmetics because melanogenesis is linked to the multiple biogenesis cascades of melanin synthesis. However, some of these compounds have side effects that may result in their restriction in the future. We report here the isolation and structural elucidation of compounds extracted from Mansonia gagei and evaluate their activity on melanogenesis inhibition. We isolated five known compounds from M. gagei and identified them as mansonone E (1), mansorin I (2), populene F (3), mansonone G (4), and mansorin B (5). After evaluating the five compounds for cytotoxicity against B16 cells and inhibitory activity on α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis, we determined that the cytotoxicity and melanogenesis-inhibitory effect of 1 were relatively low and high, respectively. Next, the effect of 1 on the expression of melanogenesis-related proteins was assessed; it was confirmed that 1 dose-dependently inhibited the expression levels of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2, cAMP response element binding protein (CREB), and microphthalmia-associated transcription factor (MITF) which were increased after stimulation by α-MSH. Furthermore, the effects of 1 on the phosphorylation levels of intracellular signaling pathway-related proteins were evaluated, and it was found that 1 dose-dependently rescued the phosphorylation of Akt and p38 mitogen-activated protein kinases (MAPK), which were up- or down-regulated after stimulation by α-MSH. In contrast, treatment with the phosphoinositide 3-kinase (PI3K)/Akt inhibitor wortmannin enhanced melanogenesis inhibition by mansonone E. Cumulatively, the data suggest that 1 suppresses α-MSH-induced melanogenesis in B16 cells by inhibiting both phosphorylation in the PI3K/Akt pathway and the expression of melanogenesis-related proteins.

The Rosiglitazone-Like Effects of Vitexilactone, a Constituent from Vitex trifolia L. in 3T3-L1 Preadipocytes.

The increased number of patients with type 2 diabetes (T2D) has become a worldwide problem, and insulin sensitizers such as thiazolidinediones (TZDs) are used as therapeutic agents. We found that extracts of Vitex trifolia L. (V. trifolia), a medicinal plant from Myanmar, induced adipogenesis similar to rosiglitazone (ROS), which is a TZD, in 3T3-L1 preadipocytes. In the present study, we attempted to isolate from V. trifolia those compounds that showed ROS-like effects. Among the extracts of hexane, ethyl acetate, and methanol obtained from V. trifolia, the ethyl acetate extract with the strongest ROS-like effects was purified by various chromatographic methods to obtain three known compounds: vitexilactone (1), vitexicarpin (2) and oleanolic acid (3). Among the isolated compounds, the ROS-like action of 1 was the strongest. The effects of 1 on 3T3-L1 cells during adipogenesis were compared with those of ROS. Both 1 and ROS increased lipid accumulation, the expression of adiponectin and GLUT4 in the cell membrane and decreased both the size of adipocytes and the phosphorylation of IRS-1, ERK1/2 and JNK in 3T3-L1 cells. In contrast, unlike ROS, the induction of proteins involved in lipogenesis was partial. ROS-like effects of 1 in 3T3-L1 cells were suppressed by the addition of bisphenol A diglycidyl ether (BADGE), one of a peroxisome proliferator-activated receptor γ (PPARγ) antagonists, suggesting that the action of 1 on adipocytes is mediated by PPARγ. From the results of the present study, it can be concluded that 1 is a novel insulin sensitizer candidate.

5,7-Dihydroxyflavone Analogues May Regulate Lipopolysaccharide-Induced Inflammatory Responses by Suppressing IκBα-Linked Akt and ERK5 Phosphorylation in RAW 264.7 Macrophages.

We studied the anti-inflammatory activity of twelve 5,7-dihydroxyflavone analogues in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. We found that chrysin (1) and 4'-methoxytricetin (9) showed relatively significant anti-inflammatory activity and low cytotoxicity. Moreover, 1 and 9 recovered the expression levels of iNOS and COX2, as well as those of the intracellular inflammatory mediators IL-1ß and IL-6, which were upregulated by LPS stimulation. In addition, 1 and 9 actively regulated the phosphorylation of IκBα, leading to the activation of NFκB. Phosphorylation of Akt and ERK5 (upstream of NFκB) by LPS stimulation was significantly regulated by 1 and 9, as well as by BIX 02189 and LY 294002, which are phosphorylation inhibitors of ERK5 and Akt, respectively. The results suggest that compounds 1 and 9 may suppress the levels of iNOS and COX2 by regulating phosphorylation of Akt, ERK5, and IκBα and thus NFκB-related signaling pathways, resulting in anti-inflammatory effects in the cells. Because 1 and 9 showed low cytotoxicity and regulated both PGE2 and NO production caused by inflammatory responses, they may hold promise as natural anti-inflammatory agents.