A series of ninety-seven diarylpentanoid derivatives were synthesized and evaluated for their anti-inflammatory activity through NO suppression assay using interferone gamma (IFN-γ)/lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Twelve compounds (9, 25, 28, 43, 63, 64, 81, 83, 84, 86, 88 and 97) exhibited greater or similar NO inhibitory activity in comparison with curcumin (14.7 ± 0.2 µM), notably compounds 88 and 97, which demonstrated the most significant NO suppression activity with IC50 values of 4.9 ± 0.3 µM and 9.6 ± 0.5 µM, respectively. A structure-activity relationship (SAR) study revealed that the presence of a hydroxyl group in both aromatic rings is critical for bioactivity of these molecules. With the exception of the polyphenolic derivatives, low electron density in ring-A and high electron density in ring-B are important for enhancing NO inhibition. Meanwhile, pharmacophore mapping showed that hydroxyl substituents at both meta- and para-positions of ring-B could be the marker for highly active diarylpentanoid derivatives.
Asthma is associated with increased pulmonary inflammation and airway hyperresponsiveness. The current use of corticosteroids in the management of asthma has recently raised issues regarding safety and lack of responsiveness in 5-10% of asthmatic individuals. The aim of the present study was to investigate the therapeutic effect of a non-steroidal small molecule that has cysteinyl leukotriene (cysLT) inhibitory activity, upon attenuation of allergic lung inflammation in an acute murine model. Mice were sensitized with ovalbumin (OVA) and treated with several intraperitoneal doses (100, 20, 2 and 0.2mg/kg) of 2,4,6,-trihydroxy-3-geranylacetophenone (tHGA). Bronchoalveolar lavage was performed, blood and lung samples were obtained and respiratory function was measured. OVA sensitization increased pulmonary inflammation and pulmonary allergic inflammation was significantly reduced at doses of 100, 20 and 2mg/kg with no effect at the lowest dose of 0.2mg/kg. The beneficial effects in the lung were associated with reduced eosinophilic infiltration and reduced secretion of Th2 cytokines and cysLTs. Peripheral blood reduction of total IgE was also a prominent feature. Treatment with tHGA significantly attenuated altered airway hyperresponsiveness as measured by the enhanced pause (Penh) response to incremental doses of methacholine. These data demonstrate that tHGA, a synthetic non-steroidal small molecule, can prevent acute allergic inflammation. This proof of concept opens further avenues of research and development of tHGA as an additional option to the current armamentarium of anti-asthma therapeutics.
A bioassay-guided investigation of Melicope ptelefolia Champ ex Benth (Rutaceae) resulted in the identification of an acyphloroglucinol, 2,4,6-trihydroxy-3-geranylacetophenone or tHGA, as the active principle inhibiting soybean 15-LOX. The anti-inflammatory action was also demonstrated on human leukocytes, where the compound showed prominent inhibitory activity against human PBML 5-LOX, with an IC(50) value of 0.42 μM, very close to the effect produced by the commonly used standard, NDGA. The compound concentration-dependently inhibited 5-LOX product synthesis, specifically inhibiting cysteinyl leukotriene LTC(4) with an IC(50) value of 1.80 μM, and showed no cell toxicity effects. The anti-inflammatory action does not seem to proceed via redox or metal chelating mechanism since the compound tested negative for these bioactivities. Further tests on cyclooxygenases indicated that the compound acts via a dual LOX/COX inhibitory mechanism, with greater selectivity for 5-LOX and COX-2 (IC(50) value of 0.40 μM). The molecular features that govern the 5-LOX inhibitory activity was thus explored using in silico docking experiments. The residues Ile 553 and Hie 252 were the most important residues in the interaction, each contributing significant energy values of -13.45 (electrostatic) and -5.40 kcal/mol (electrostatic and Van der Waals), respectively. The hydroxyl group of the phloroglucinol core of the compound forms a 2.56Å hydrogen bond with the side chain of the carboxylate group of Ile 553. Both Ile 553 and Hie 252 are crucial amino acid residues which chelate with the metal ion in the active site. Distorting the geometry of these ligands could be the reason for the inhibition activity shown by tHGA. The molecular simulation studies supported the bioassay results and served as a good model for understanding the way tHGA binds in the active site of human 5-LOX enzyme.
The anti-inflammatory activity of zerumbone (1), a natural cyclic sesquiterpene isolated from Zingiber zerumbet Smith was investigated using carrageenan-induced paw edema and cotton pellet-induced granuloma tissue formation test in mice. It was demonstrated that intraperitoneal administration of 1 at a dose of 5, 10, 50 and 100 mg/kg produced significant dose-dependent inhibition of paw edema induced by carrageenan. It was also demonstrated that 1 at similar doses significantly suppressed granulomatous tissue formation in cotton pellet-induced granuloma test.
The present study examined the potential antinociceptive activity of flavokawin B (6'-hydroxy-2',4'-dimethoxychalcone), a synthetic chalcone using chemical- and thermal-induced nociception models in mice. It was demonstrated that flavokawin B (FKB; 0.3, 1, 3 and 10 mg/kg) administered via both oral (p.o.) and intraperitoneal (i.p.) routes produced significant and dose-dependent inhibition in the abdominal constrictions induced by acetic acid, with the i.p. route producing antinociception of approximately 7-fold more potent than the p.o. route. It was also demonstrated that FKB produced significant inhibition in the two phases of the formalin-induced paw licking test. In addition, the same treatment of flavokawin B (FKB) exhibited significant inhibition of the neurogenic nociceptive induced by intraplantar injections of glutamate and capsaicin. Likewise, this compound also induced a significant increase in the response latency period to thermal stimuli in the hot plate test and its antinociceptive effect was not related to muscle relaxant or sedative action. Moreover, the antinociception effect of the FKB in the formalin-induced paw licking test and the hot plate test was not affected by pretreatment of non-selective opioid receptor antagonist, naloxone. The present results indicate that FKB produced pronounced antinociception effect against both chemical and thermal models of pain in mice that exhibited both peripheral and central analgesic activity.
Curcumin is a highly pleiotropic molecule with significant regulatory effects upon inflammation and inflammatory related diseases. However curcumin has one major important limitation in which it has poor bioavailability. Design of synthetic structural derivatives of curcumin is but one approach that has been used to overcome its poor bioavailability while retaining, or further enhancing, its drug-like effects. We have synthesized a series of curcumin analogues and describe the effects of 2,6-bis-4-(hydroxyl-3-methoxy-benzylidine)-cyclohexanone or BHMC upon nitric oxide and cytokine synthesis in cellular models of inflammation. BHMC showed a significant dose-response inhibitory action upon the synthesis of NO and we have shown that this effect was due to suppression of both iNOS gene and enzyme expression without any effects upon scavenging of nitrite. We also demonstrated that BHMC has a very minimal effect upon iNOS activity with no effect at all upon the secretion of PGE(2) but has a strong inhibitory effect upon MCP-1 and IL-10 secretion and gene expression. Secretion and gene expression of TNF-alpha and IL-6 were moderately inhibited whereas IL-8 and IL-1beta were not altered. We conclude that BHMC selectively inhibits the synthesis of several inflammatory mediators. BHMC should be considered a promising drug lead for preclinical and further pharmacological studies.
Some chalcones, such as hydroxychalcones have been reported previously to inhibit major pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E(2) (PGE(2)), tumor necrosis factor-alpha (TNF-alpha) and reactive oxygen species production by suppressing inducible enzyme expression via inhibition of the mitogen-activated protein kinase (MAPK) pathway and nuclear translocation of critical transcription factors. In this report, the effects of cardamonin (2',4'-dihydroxy-6'-methoxychalcone), a chalcone that we have previously isolated from Alpinia rafflesiana, was evaluated upon two cellular systems that are repeatedly used in the analysis of anti-inflammatory bioactive compounds namely RAW 264.7 cells and whole blood. Cardamonin inhibited NO and PGE(2) production from lipopolysaccharide- and interferon-gamma-induced RAW cells and whole blood with IC(50) values of 11.4 microM and 26.8 microM, respectively. Analysis of thromboxane B(2) (TxB(2)) secretion from whole blood either stimulated via the COX-1 or COX-2 pathway revealed that cardamonin inhibits the generation of TxB(2) via both pathways with IC(50) values of 2.9 and 1.1 microM, respectively. Analysis of IC(50) ratios determined that cardamonin was more COX-2 selective in its inhibition of TxB(2) with a ratio of 0.39. Cardamonin also inhibited the generation of intracellular reactive oxygen species and secretion of TNF-alpha from RAW 264.7 cells in a dose responsive manner with IC(50) values of 12.8 microM and 4.6 microM, respectively. However, cardamonin was a moderate inhibitor of lipoxygenase activity when tested in an enzymatic assay system, in which not a single concentration tested was able to cause an inhibition of more than 50%. Our results suggest that cardamonin acts upon major pro-inflammatory mediators in a similar fashion as described by previous work on other closely related synthetic hydroxychalcones and strengthens the conclusion of the importance of the methoxyl moiety substitution on the 4' or 6' locations of the A benzene ring.
In search of potent anti-inflammatory agents, twenty-four chalcone derivatives including seven new compounds (13 - 17, 21 and 23) containing pyrrole moiety were designed, synthesized, and assessed for their nitric oxide (NO) and prostaglandin E2 (PGE2) suppression ability on IFN-γ/LPS-induced RAW 264.7 macrophage cells. Results showed that none of the synthesized compounds were PAINS-associated molecules, with 3-(2,5-dimethoxyphenyl)-1-(1H-pyrrol-2-yl)-prop-2-en-1-one (compound 16) exhibiting remarkable inhibition activity towards PGE2 and NO production with IC50 values of 0.5 ± 1.5 µM and 12.1 ± 1.5 µM, respectively. Physicochemical and ADMET studies showed that majority of the compounds obey to Lipinski's rule of five (RO5) having high blood brain barrier (BBB) penetration, human intestinal absorption (HIA), P- glycoprotein (PgP) inhibition and plasma binding protein (PPB) inhibition. The obtained atomic coordinates for the single-crystal XRD of 16 were then applied in a molecular docking simulation, and compound 16 was found to participate in a number of important binding interactions in the binding sites of ERK and mPGES-1. Based on these results, we have observed the potential of compound 16 as a new hit anti-inflammatory agent, and these findings could serve as a basis for further studies on its mechanism of action.
A series of twenty-four 2-benzoyl-6-benzylidenecyclohexanone analogs were synthesized and evaluated for their nitric oxide inhibition and antioxidant activity. Six compounds (3, 8, 10, 17, 18 and 19) were found to exhibit significant NO inhibitory activity in LPS/IFN-induced RAW 264.7 macrophages, of which compound 10 demonstrated the highest activity with the IC50 value of 4.2 ± 0.2 μM. Furthermore, two compounds (10 and 17) displayed antioxidant activity upon both the DPPH scavenging and FRAP analyses. However, none of the 2-benzoyl-6-benzylidenecyclohexanone analogs significantly scavenged NO radical. Structure-activity comparison suggested that 3,4-dihydroxylphenyl ring is crucial for bioactivities of the 2-benzoyl-6-benzylidenecyclohexanone analogs. The results from this study and the reports from previous studies indicated that compound 10 could be a candidate for further investigation on its potential as a new anti-inflammatory agent.
A new series of pyrazole, phenylpyrazole, and pyrazoline analogs of diarylpentanoids (excluding compounds 3a, 4a, 5a, and 5b) was pan-assay interference compounds-filtered and synthesized via the reaction of diarylpentanoids with hydrazine monohydrate and phenylhydrazine. Each analog was evaluated for its anti-inflammatory ability via the suppression of nitric oxide (NO) on IFN-γ/LPS-activated RAW264.7 macrophage cells. The compounds were also investigated for their inhibitory capability toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), using a modification of Ellman's spectrophotometric method. The most potent NO inhibitor was found to be phenylpyrazole analog 4c, followed by 4e, when compared with curcumin. In contrast, pyrazole 3a and pyrazoline 5a were found to be the most selective and effective BChE inhibitors over AChE. The data collected from the single-crystal X-ray diffraction analysis of compound 5a were then applied in a docking simulation to determine the potential binding interactions that were responsible for the anti-BChE activity. The results obtained signify the potential of these pyrazole and pyrazoline scaffolds to be developed as therapeutic agents against inflammatory conditions and Alzheimer's disease.
Limited tumor penetrability of anti-cancer drugs is recognized as one of the major factors that lead to poor anti-tumor activity. SRJ09 (3,19-(2-bromobenzylidene) andrographolide) has been identified as a lead anti-cancer agent for colon cancer. Recently, this compound was shown by us to be a mutant K-Ras binder. In this present study, the penetrability of SRJ09 through the DLD-1 colon cancer multicell layer (MCL) was evaluated. The amount of SRJ09 that penetrated through the MCL was quantitated by utilizing high performance liquid chromatography (HPLC). Histopathological staining was used to visualize the morphology of MCL. A chemosensitivity assay was performed to assess the anti-cancer activity of SRJ09 in DLD-1 cells. SRJ09 was able to penetrate through DLD-1 MCL and is inversely proportional with the MCL thickness. The flow rates for SRJ09 through MCL were 0.90 ± 0.20 μM/min/cm(2) and 0.56 ± 0.06 μM/min/cm(2) for days 1 and 5, respectively, which are better than doxorubicin. Histopathological examination revealed that the integrity of the DLD-1 MCL was retained and no visible damage was inflicted on the cell membrane, confirming the penetration of SRJ09 was by diffusion. Short term exposure (1 h) in DLD-1 cells demonstrated SRJ09 had IC50 of 41 μM which was approximately 4-folds lower than andrographolide, the parent compound of SRJ09. In conclusion, SRJ09 successfully penetrated through DLD-1 MCL by diffusion and emerged as a potential candidate to be developed as a clinically viable anti-colon cancer drug.
Phytochemical investigation on the leaves of Labisia pumila (Myrsinaceae), an important medicinal herb in Malaysia, has led to the isolation of 1-O-methyl-6-acetoxy-5-(pentadec-10Z-enyl)resorcinol (1), labisiaquinone A (2) and labisiaquinone B (3). Along with these, 16 known compounds including 1-O-methyl-6-acetoxy-5-pentadecylresorcinol (4), 5-(pentadec-10Z-enyl)resorcinol (5), 5-(pentadecyl)resorcinol (6), (-)-loliolide (7), stigmasterol (8), 4-hydroxyphenylethylamine (9), 3,4,5-trihydroxybenzoic acid (10), 3,4-dihydroxybenzoic acid (11), (+)-catechin (12), (-)-epicatechin (13), kaempferol-3-O-α-rhamnopyranosyl-7-O-β-glycopyranoside (14), kaempferol-4'-O-β-glycopyranoside (15), quercetin-3-O-α-rhamnopyranoside (16), kaempferol-3-O-α-rhamnopyranoside (17), (9Z,12Z)-octadeca-9,12-dienoic acid (18) and stigmasterol-3-O-β-glycopyranoside (19) were also isolated. The structures of these compounds were established on the basis of 1D and 2D NMR spectroscopy techniques (¹H, ¹³C, COSY, HSQC, NOESY and HMBC experiments), mass spectrometry and chemical derivatization. Among the constituents tested 1 and 4 exhibited strongest cytotoxic activity against the PC3, HCT116 and MCF-7 cell lines (IC₅₀ values ≤ 10 μM), and they showed selectivity towards the first two-cell lines relative to the last one.
Our preliminary screening has shown that curcumin derivative BDMC33 [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] exerted promising nitric oxide inhibitory activity in activated macrophages. However, the molecular basis and mechanism for its pharmacological action is yet to be elucidated. The aim of this study was to investigate the anti-inflammatory properties of BDMC33 and elucidate its underlying mechanism action in macrophage cells. Our current study demonstrated that BDMC33 inhibits the secretion of major pro-inflammatory mediators in stimulated macrophages, and includes NO, TNF-α and IL-1β through interference in both nuclear factor kappaB (NF-κB) and mitogen activator protein kinase (MAPK) signaling cascade in IFN-γ/LPS-stimulated macrophages. Moreover, BDMC33 also interrupted LPS signaling through inhibiting the surface expression of CD-14 accessory molecules. In addition, the inhibitory action of BDMC33 not only restricted the macrophages cell (RAW264.7), but also inhibited the secretion of NO and TNF-α in IFN-γ/LPS-challenged microglial cells (BV-2). The experimental data suggests the inflammatory action of BDMC33 on activated macrophage-like cellular systems, which could be used as a future therapeutic agent in the management of chronic inflammatory diseases.
We previously showed that 2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone (BHMC), suppressed the synthesis of various proinflammatory mediators. In this study we explain the mechanism of action of BHMC in lipopolysaccharide (LPS)-induced U937 monocytes and further show that BHMC prevents lethality of CLP-induced sepsis. BHMC showed dose-dependent inhibitory effects on p38, JNK and ERK 1/2 activity as determined by inhibition of phosphorylation of downstream transcription factors ATF-2, c-Jun and Elk-1 respectively. Inhibition of these transcription factors subsequently caused total abolishment of AP-1-DNA binding. BHMC inhibited p65 NF-κB nuclear translocation and DNA binding of p65 NF-κB only at the highest concentration used (12.5μM) but failed to alter phosphorylation of JNK, ERK1/2 and STAT-1. Since the inhibition of p38 activity was more pronounced we evaluated the possibility that BHMC may bind to p38. Molecular docking experiments confirmed that BHMC fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We also show that BHMC was able to improve survival from lethal sepsis in a murine caecal-ligation and puncture (CLP) model.
In a preliminary screen, Aaptos aaptos showed significant cytotoxic activity towards a panel of cell lines and was thus subjected to bioassay-guided isolation of the bioactive constituents. In addition to the known aaptamine, two new derivatives of the alkaloid were isolated from the bioactive chloroform fraction of the crude methanolic extract. Detailed analysis by NMR and mass spectroscopy enabled their identification to be 3-(phenethylamino)demethyl(oxy)aaptamine and 3-(isopentylamino)demethyl(oxy) aaptamine. The cytotoxic activities of the three alkaloids were further evaluated against CEM-SS cells.
The plant Andrographis paniculata found throughout Southeast Asia contains Andrographolide 1, a diterpenoid lactone, which has antitumour activities against in vitro and in vivo breast cancer models. In the present study, we report on the synthesis of andrographolide derivatives, 3,19-isopropylideneandrographolide (2), 14-acetyl-3,19-isopropylideneandrographolide (3) and 14-acetylandrographolide (4), and their in vitro antitumour activities against a 2-cell line panel consisting of MCF-7 (breast cancer cell line) and HCT-116 (colon cancer cell line). Compounds 2 and 4 were also screened at the US National Cancer Institute (NCI) for their activities against a panel of 60 human cancer cell lines derived from nine cancer types. Compound 2 was found to be selective towards leukaemia and colon cancer cells, and compound 4 was selective towards leukaemia, ovarian and renal cancer cells at all the dose-response parameters. Compounds 2 and 4 showed non-specific phase of the cell cycle arrest in MCF-7 cells treated at different intervals with different concentrations. NCI's COMPARE and SOM mechanistic analyses indicated that the anticancer activities of these new class of compounds were not similar to that of standard anticancer agents, suggesting novel mechanism(s) of action.
Andrographolide (AGP) and 14-deoxy-11,12-didehydroandrographolide (DDAG), two main diterpenoid constituents of Andrographis paniculata were previously shown to ameliorate asthmatic symptoms in a mouse model. However, due to inadequacies of both compounds in terms of drug-likeness, DDAG analogues were semisynthesised for assessment of their anti-asthma activity. A selected analogue, 3,19-diacetyl-14-deoxy-11,12-didehydroandrographolide (SRS27), was tested for inhibitory activity of NF-κB activation in TNF-α-induced A549 cells and was subsequently evaluated in a mouse model of ovalbumin (OVA)-induced asthma. Female BALB/c mice, 6-8weeks old were sensitized on days 0 and 14, and challenged on days 22, 23 and 24 with OVA. Compound or vehicle (3% dimethyl sulfoxide) was administered intraperitoneally 1h before and 11h after each OVA aerosol challenge. On day 25, pulmonary eosinophilia, airway hyperresponsiveness, mucus hypersecretion, inflammatory cytokines such as IL-4, -5 and -13 in BAL fluid, gene expression of inflammatory mediators such as 5-LOX, E-selectin, VCAM-1, CCL5, TNF-α, AMCase, Ym2, YKL-40, Muc5ac, CCL2 and iNOS in animal lung tissues, and serum IgE were determined. SRS27 at 30μM was found to suppress NF-κB nuclear translocation in A549 cells. In the ovalbumin-induced mouse asthma model, SRS27 at 3mg/kg displayed a substantial decrease in pulmonary eosinophilia, BAL fluid inflammatory cytokines level, serum IgE production, mucus hypersecretion and gene expression of inflammatory mediators in lung tissues. SRS27 is the first known DDAG analogue effective in ameliorating inflammation and airway hyperresponsiveness in the ovalbumin-induced mouse asthma model.
Diabetes mellitus (DM) is a chronic disease that can affect metabolism of glucose and other metabolites. In this study, the normal- and obese-diabetic rats were compared to understand the diabetes disorders of type 1 and 2 diabetes mellitus. This was done by evaluating their urine metabolites using proton nuclear magnetic resonance (1H NMR)-based metabolomics and comparing with controls at different time points, considering the induction periods of obesity and diabetes. The biochemical parameters of the serum were also investigated. The obese-diabetic model was developed by feeding the rats a high-fat diet and inducing diabetic conditions with a low dose of streptozotocin (STZ) (25 mg/kg bw). However, the normal rats were induced by a high dose of STZ (55 mg/kg bw). A partial least squares discriminant analysis (PLS-DA) model showed the biomarkers of both DM types compared to control. The synthesis and degradation of ketone bodies, tricarboxylic (TCA) cycles, and amino acid pathways were the ones most involved in the variation with the highest impact. The diabetic groups also exhibited a noticeable increase in the plasma glucose level and lipid profile disorders compared to the control. There was also an increase in the plasma cholesterol and low-density lipoprotein (LDL) levels and a decline in the high-density lipoprotein (HDL) of diabetic rats. The normal-diabetic rats exhibited the highest effect of all parameters compared to the obese-diabetic rats in the advancement of the DM period. This finding can build a platform to understand the metabolic and biochemical complications of both types of DM and can generate ideas for finding targeted drugs.
The methanol extract of the dried ripe fruits of Alpinia rafflesiana was investigated for its DPPH free radical scavenger constituents. 2',3',4',6'-Tetrahydroxychalcone (7), which has never been isolated from natural sources was found to be most active as a DPPH free radical scavenger with the IC50 value of 55 microM. Other known compounds isolated from this species include 5,6-dehydrokawain (1), flavokawin B (2). 1,7-diphenyl-5-hydroxy-6-hepten-3-one (3), (-)-pinocembrin (4), cardamonin (5) and (-)-pinostrobin (6). The DPPH free radical scavenger compounds were detected using TLC autographic analysis. The percentage inhibition of DPPH free radical scavenging activity was measured on isolates (5-7) using colorimetric analysis.
We previously showed that 3-(2-hydroxyphenyl)-1-(5-methyl-furan-2-y-l)propenone (HMP), suppressed the synthesis of various proinflammatory mediators. In this study, HMP showed a dose-dependent inhibition of NO synthesis in the RAW 264.7 murine macrophage line. The inhibition of NO synthesis was related to inhibition of p38 phosphorylation and kinase activity that led to significant inhibition of phosphorylation of ATF-2. This effect in turn caused inhibition of AP-1-DNA binding which partially explains the inhibitory effect upon the synthesis of iNOS. HMP had no effect upon phosphorylation of JNK, ERK1/2 and STAT-1. Kinase activity of JNK and ERK1/2 was also not affected by HMP as determined by levels of phosphorylated c-jun and phosphorylated elk-1. Furthermore HMP failed to block phosphorylation of IκBα, and subsequent nuclear translocation and DNA-binding activity of p65 NF-κB in IFN-γ/LPS-induced RAW 264.7 cells. Molecular docking experiments confirmed that HMP fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We conclude that the synthetic HMP is a chalcone analogue that selectively inhibits the p38/ATF-2 and AP-1 signaling pathways in the NO synthesis by the macrophage RAW 264.7.