Apoptotic Potential of Iloneoside from Gongronema latifolium Benth against Prostate Cancer Cells Using In Vitro and In Silico Approach.

Prostate cancer is a major cause of cancer-related mortality in men worldwide. The anti-proliferative activity of Gongronema latifolium leaf extracts on some cancer cells has been reported. Herein, we investigated the growth inhibitory effect of the Gongronema latilolium leaf methanol extract and isolated pregnane (iloneoside) against prostate cancer cell lines using the MTT cell proliferation assay, apoptosis quantification, cell cycle analysis using flow cytometry and computational analysis molecular docking, molecular dynamics simulation (MDs), binding free energy computation and cluster analysis. In addition, UPLC-ESI-TOFMS chemical fingerprinting of previously isolated compounds was performed. The extract inhibited the growth of the cell lines with an IC50 of 49.3 µg/ml and 28.4 µg/ml for 24 h and 48 h, respectively, for PC3; and 43.7 µg/ml and 22.3 µg/ml for 24 h and 48 h, respectively, for DU145. Iloneoside demonstrated low inhibitory activities against PC3 and DU145 (IC50 > 80 µM). Apoptotic quantification and cell cycle analysis further showed that iloneoside induced apoptosis in a few cells at a dose of 200 uM. The ensemble-based molecular docking of the iloneoside to BCL-XL and BCL-2 proteins, and docking to MCL-1, BCL-A1 and BFL-1 proteins, respectively, presented binding energies of -7.22 ± 0.5, -8.12 ± 0.55, -7.1, -7.2 and -6.3 kcal/mol, while the MM/PBSA binding free energy was -25.72 ± 7.22 and -27.76 ± 11.32 kcal/mol for BCL-XL and BCL-2 proteins. Furthermore, iloneoside was stable during the 100 ns MDs analysis, while the clustering of the MDs trajectories showed that the interactions were strongly preserved. Iloneoside, in part, or in synergy with other constituents, may be responsible for the antiproliferative activities of the leaf, subject to further investigation.

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.

Comparative analysis of p-terphenylquinone and seriniquinone derivatives as reactive oxygen species-modulating agents.

Quinones are widespread in plants, animals, insects, and microorganisms. Several anticancer agents contain quinone structures as critical parts to show remarkable potential and distinctive modes of actions. The purpose of this study was to investigate the structure-activity relationships of microbial quinones and their derivatives as anticancer agents. A series of p-terphenylquinone and seriniquinone derivatives were therefore prepared. Treatment of the synthesized quinones possessed antiproliferative activity on human leukemia HL-60 cells in a dose-dependent fashion. In addition, seriniquinone derivatives elevated cellular reactive oxygen species (ROS) levels, thereby triggering the ensuing apoptotic events. Our findings emphasize the excellent potential of seriniquinone derivatives as redox cycling-induced ROS-modulating anticancer agents.

Diversity of Adenostemma lavenia, multi-potential herbs, and its kaurenoic acid composition between Japan and Taiwan.

Adenostemma lavenia (L.) Kuntze (Asteraceae) is widely distributed in tropical regions of East Asia, and both A. lavenia and A. madurense (DC) are distributed in Japan. In China and Taiwan, A. lavenia is used as a folk medicine for treating lung congestion, pneumonia, and hepatitis. However, neither phylogenic nor biochemical analysis of this plants has been performed to date. We have reported that the aqueous extract of Japanese A. lavenia contained high levels of ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11αOH-KA; a kaurenoic acid), which is a potent anti-melanogenic compound. Comparison of chloroplast DNA sequences suggested that A. lavenia is originated from A. madurense. Analyses of kaurenoic acids revealed that Japanese A. lavenia and A. madurense contained high levels of 11αOH-KA and moderate levels of 11α,15OH-KA, while Taiwanese A. lavenia mainly contained 9,11αOH-KA. The diverse biological activities (downregulation of Tyr, tyrosinase, gene expression [anti-melanogenic] and iNOS, inducible nitric oxide synthase, gene expression [anti-inflammatory], and upregulation of HO-1, heme-oxygenase, gene expression [anti-oxidative]) were associated with 11αOH-KA and 9,11αOH-KA but not with 11α,15OH-KA. Additionally, 11αOH-KA and 9,11αOH-KA decreased Keap1 (Kelch-like ECH-associated protein 1) protein levels, which was accompanied by upregulation of protein level and transcriptional activity of Nrf2 (NF-E2-related factor-2) followed by HO-1 gene expression. 11αOH-KA and 9,11αOH-KA differ from 11α,15OH-KA in terms of the presence of a ketone (αß-unsaturated carbonyl group, a thiol modulator) at the 15th position; therefore, thiol moieties on the target proteins, including Keap1, may be important for the biological activities of 11αOH-KA and 9,11αOH-KA and A. lavenia extract.

Review of sialic acid's biochemistry, sources, extraction and functions with special reference to edible bird's nest.

Sialic acids are a group of nine-carbon α-keto acids. Sialic acid exists in more than 50 forms, with the natural types discovered as N-acetylneuraminic acid (Neu5Ac), deaminoneuraminic acid (2-keto-3-deoxy-nonulononic acid or Kdn), and N-glycolylneuraminic acid (Neu5Gc). Sialic acid level varies depending on the source, where edible bird's nest (EBN), predominantly Neu5Ac, is among the major sources of sialic acid. Due to its high nutritive value and complexity, sialic acid has been studied extensively through acid, aqueous, and enzymatic extraction. Although detection by chromatographic methods or mass spectrometry is common, the isolation and recovery work remained limited. Sialic acid is well-recognised for its bioactivities, including brain and cognition development, immune-enhancing, anti-hypertensive, anticancer, and skin whitening properties. Therefore, sialic acid can be used as a functional ingredient in the various industries. This paper reviews the current trend in the biochemistry, sources, extraction, and functions of sialic acids with special reference to EBN.

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.

Citrulluside H and citrulluside T from young watermelon fruit attenuate ultraviolet B radiation-induced matrix metalloproteinase expression through the scavenging of generated reactive oxygen species in human dermal fibroblasts.

BACKGROUND: Prolonged exposure to UV radiation disrupts the oxidative stress defense systems; in the absence of adequate reactive oxygen species (ROS)-scavenging mechanisms, the ROS produced cause oxidative damage to DNA, proteins, carbohydrates, and lipids. Thus, ROS elimination can protect against photoaging and possibly skin cancer. We recently isolated two novel phenolic glycosides (citrulluside H and citrulluside T) with antioxidative activity from young watermelon (Citrullus lanatus) fruits. In the present study, we evaluated the protective effect of these compounds against UV-B-induced photodamage in normal human dermal fibroblasts (NHDFs). METHODS: NHDFs were exposed to UV-B irradiation at 25 mJ/cm2 . Matrix metalloproteinase (MMP)-1 and MMP-3 mRNA levels were measured by quantitative real-time PCR. MMP protein expression, as well as MAPK and NF-κB signaling, was investigated by Western blot analysis. Activation protein-1 (AP-1) transcriptional activity was determined using a luciferase reporter assay. Intracellular ROS levels were measured using the CellROX™ Deep Red Reagent. RESULTS: Citrulluside H and citrulluside T significantly downregulated the protein and mRNA expression of MMP-1 and MMP-3, which are associated with the onset of skin wrinkle formation. Importantly, inhibition of UV-B-induced MMP expression in NHDFs by citrulluside H and citrulluside T was primarily due to the attenuation of MAPK/AP-1 and NF-κB signaling via scavenging of the generated ROS. CONCLUSION: Our findings delineated both citrulluside H and citrulluside T as potential agents for alleviating UV-induced skin photoaging.

A New Depsidone from Teloschistes flavicans and the Antileukemic Activity.

Lichens produce a variety of secondary metabolites that could be potential sources of pharmaceutically useful chemicals. However, only a limited number of lichen metabolites have been investigated for their biological significance. The objective of this study was to identify the potential compounds responsible for the antileukemic activity of lichen Teloschistes flavicans. Among three fractions (n-hexane, EtOAc, and MeOH-H2O), the ethyl acetate (EtOAc) fraction of T. flavicans methanolic extract showed the strongest inhibition in the HL-60 cell line. Additionally, the EtOAc fraction was further purified to obtain a new depsidone, 2,7-dichloro-3,8-dimethoxy-1,6,9-trimethyl-11H-dibenzo[b,e][1,4]dioxepin-11-one, named as flavicansone, along with rhizonic acid, parietin, and vicanicin. Flavicansone demonstrated the most significant inhibitory action against cell proliferation among the four isolated compounds.

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.

Clerodendrum volubile Ethanol Leaf Extract: A Potential Antidote to Doxorubicin-Induced Cardiotoxicity in Rats.

Doxorubicin is widely applied in hematological and solid tumor treatment but limited by its off-target cardiotoxicity. Thus, cardioprotective potential and mechanism(s) of CVE in DOX-induced cardiotoxicity were investigated using cardiac and oxidative stress markers and histopathological endpoints. 50-400 mg/kg/day CVE in 5% DMSO in distilled water were investigated in Wistar rats intraperitoneally injected with 2.5 mg/kg DOX on alternate days for 14 days, using serum troponin I and LDH, complete lipid profile, cardiac tissue oxidative stress marker assays, and histopathological examination of DOX-treated cardiac tissue. Preliminary qualitative and quantitative assays of CVE's secondary metabolites were also conducted. Phytochemical analyses revealed the presence of flavonoids (34.79 ± 0.37 mg/100 mg dry extract), alkaloids (36.73 ± 0.27 mg/100 mg dry extract), reducing sugars (07.78 ± 0.09 mg/100 mg dry extract), and cardiac glycosides (24.55 ± 0.12 mg/100 mg dry extract). 50-400 mg/kg/day CVE significantly attenuated increases in the serum LDH and troponin I levels. Similarly, the CVE dose unrelatedly decreased serum TG and VLDL-c levels without significant alterations in the serum TC, HDL-c, and LDL-c levels. Also, CVE profoundly attenuated alterations in the cardiac tissue oxidative stress markers' activities while improving DOX-associated cardiac histological lesions that were possibly mediated via free radical scavenging and/or antioxidant mechanisms. Overall, CVE may play a significant therapeutic role in the management of DOX-induced cardiotoxicity in humans.

The High Content of Ent-11α-hydroxy-15-oxo-kaur- 16-en-19-oic Acid in Adenostemma lavenia (L.) O. Kuntze Leaf Extract: With Preliminary in Vivo Assays.

BACKGROUND: Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11αOH-KA) is a multifunctional biochemical found in some ferns, Pteris semipinnata, and its congeneric species. Although a number of therapeutic applications of 11αOH-KA have been proposed (e.g., anti-cancer, anti-inflammation, and skin whitening), the content of 11αOH-KA in these ferns is not high. Adenostemma lavenia (L.) O. Kuntze, an Asteraceae, has also been reported to contain 11αOH-KA. The decoction (hot water extract) of whole plants of A. lavenia is used as a folk remedy for inflammatory disorders, such as hepatitis and pneumonia, suggesting that 11αOH-KA may be the ingredient responsible for the medicinal properties of this plant. METHODS: The anti-melanogenic activities of the water extracts of A. lavenia leaves and Pteris dispar Kunze (a cognate of P. semipinnata) leaves were compared in mouse B16F10 melanoma cells. The amount of 11αOH-KA was measured by using liquid chromatography spectrometry. C57BL/6J mice were treated with the water extract of A. lavenia leaf, and the blood concentration of 11αOH-KA was measured. The in vivo efficacy of the water extract of A. lavenia leaf was evaluated according to tis anti-melanogenic activity by monitoring hair color. RESULTS: Although both the extracts (A. lavenia and P. dispar Kunze) showed high anti-melanogenic activities, only A. lavenia contained a high amount of 11αOH-KA, approximately 2.5% of the dry leaf weight. 11αOH-KA can be purified from A. lavenia leaves in two steps: water extraction followed by chloroform distribution. The treatment of mice with the water extract of A. lavenia leaf suppresses pigmentation in their hairs. CONCLUSIONS: Despite the small number of mice examined, the present preliminary result of the suppressed hair pigmentation suggests that the water extract of A. lavenia leaf and the ingredient that is possibly responsible for this-11αOH-KA-are new materials for oral cosmetics. The results may also be helpful in the future development of functional foods and methods to treat patients suffering from hyperpigmentation disorders, such as melasma.

Polyalthia longifolia Extract Triggers ER Stress in Prostate Cancer Cells Concomitant with Induction of Apoptosis: Insights from In Vitro and In Vivo Studies.

Plant-based therapies are being explored to prevent or treat several cancer types. The antioxidant properties of Polyalthia longifolia plant are well established. In our previous work, we demonstrated the presence of cytotoxic compounds in the methanol extract of Polyalthia longifolia (MEP) with potent activity against human leukemia cells. In the present study, we evaluated the efficacy of MEP against prostate cancer (PCa) and established the molecular basis of its effect in in vitro and in vivo models. We observed that MEP treatment resulted in a significant decrease in the growth and viability of PCa cells, associated with arrest in the G1/S phase of the cell cycle. Apoptosis was confirmed as the primary mode of MEP-induced cell death through activation of the intrinsic apoptotic machinery. Proteomic and biochemical studies identified BiP as an important target of MEP with the activation of the ER stress pathway, as a potential mechanism driving MEP-induced apoptosis. The extract exhibited strong efficacy in the PCa xenograft mouse model with significant inhibition of tumor growth and reduced tumor burden. Taken together, our findings indicate that MEP-induced apoptosis in PCa cells concomitant with the activation of the ER stress pathways results in the inhibition of tumor growth, in vitro and in vivo. Our studies provide initial evidence of the efficacy of MEP against PCa and advocate for in-depth studies in other preclinical models for its possible use in clinical settings.

Preparation of Monoacylglycerol Derivatives from Indonesian Edible Oil and Their Antimicrobial Assay against Staphylococcus aureus and Escherichia coli.

In the present work, linoleic acid and oleic acid were isolated from Indonesian corn oil and palm oil and they were used to prepare monoacylglycerol derivatives as the antibacterial agent. Indonesian corn oil contains 57.74% linoleic acid, 19.88% palmitic acid, 11.84% oleic acid and 3.02% stearic acid. While Indonesian palm oil contains 44.72% oleic acid, 39.28% palmitic acid, 4.56% stearic acid and 1.54% myristic acid. The oleic acid was purified by using Urea Inclusion Complex (UIC) method and its purity was significantly increased from 44.72% to 94.71%. Meanwhile, with the UIC method, the purity of ethyl linoleate was increased from 57.74% to 72.14%. 1-Monolinolein and 2-monoolein compounds were synthesized via two-step process from the isolated linoleic acid and oleic acid, respectively. The preliminary antibacterial assay shows that the 1-monolinolein did not give any antibacterial activity against Staphylococcus aureus and Escherichia coli, while 2-monoolein showed weak antibacterial activity against Staphylococcus aureus.

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.

Iron-Promoted Intramolecular Cascade Cyclization for the Synthesis of Selenophene-Fused, Quinoline-Based Heteroacenes.

Herein, we report the Fe(III)-promoted linear intramolecular cascade cyclization of 1,3-diyne and 1,3,5-triyne for the construction of selenophene-fused, quinoline-based heteroacene scaffolds. In one step, 1,3-diyne and 1,3,5-triyne were cyclized via diversified internal nucleophiles by using diorganyl diselenides. The diorganyl diselenide plays dual role, one as a cyclizing agent and second as insertion of one and/or two selenium atom and one R'-Se group in the final product. This is highly important in terms of atom economy. Diversified internal nucleophiles were used to afford quinoline- and acridine-based cores. The synthesized selenophene-fused derivatives showed λmax, Fmax, and Φf values in the range from 370-411 nm, 427-472 nm, and 0.003-0.059, respectively, in dichloromethane solvent.

Synthesis of carbazoloquinone derivatives and their antileukemic activity via modulating cellular reactive oxygen species.

Carbazoloquinone alkaloids are of great interest as privileged structures for anticancer drug molecules. The purpose of this study was to investigate the structure-activity relationships of carbazoloquinone derivatives as anticancer agents. A series of carbazoloquinones including murrayaquinone A, koeniginequinones A and B, and related analogues were therefore prepared. Palladium-catalyzed intramolecular cyclization reaction mechanism was well elucidated by DFT calculations. Treatment of the synthesized derivatives showed cytotoxicity on human leukemia HL-60 cells in a dose-dependent fashion. In addition, murrayaquinone A and ß-brazanquinone elevated cellular levels of reactive oxygen species (ROS), thereby triggering apoptosis. Our findings emphasize the excellent potential of carbazoloquinone derivatives as ROS-inducing anticancer agents.

Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells.

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.