Anti-inflammatory Screening Research Articles

Optimization of an anti-inflammatory screening model on the RAW 264.7 macrophage cell

Inflammation is the response of living tissues to the injury. Prolonged or inappropriate inflammation has been involved with many diseases such as, cancer, diabetes, heart disease… These days, inflammation has been treated by nonsteroidal and steroidal anti-inflammatory drugs, which have a lot of side effects. It opens the need and interest of new drugs discovery. Particularly, scientific and pharmaceutical communities show great interest in finding new anti-inflammatory compounds in traditional medicinal plants. This study aimed to optimize an in vitro anti-inflammatory model. Cell heterogeneity, cell density, LPS concentration and LPS incubation time were chosen to optimize the production of nitric oxide (NO) in RAW 264.7 macrophage cells. Our results show that 104 cells/well, FBS 1 %, starvation for 6 h, LPS 0.5 µg/mL in 24 h were optimized parameters in the model. Then, extracts from Hedyotis capitellata, a traditional medicinal plant used by K’ho minority, Bidoup Nuiba national park, Lam Dong province, Vietnam, was chosen to evaluate the in vitro antiinflammatory model. The anti-inflammatory activity was tested by measuring the production of NO in lipopolysaccharide-activated RAW 264.7 macrophage cells. The experimental data indicate that the extracts of this plant decreased NO production in LPS-stimulated RAW 264.7, particularly, the petroleum ether fraction at the concentration of 23.8 µg/mL inhibited NO production by 128.20 %; whereas dexamethasone 50 µM inhibited NO accumulation to 112 %.

New benzimidazothiazole derivatives as anti-inflammatory, antitumor active agents: Synthesis, in-vitro and in-vivo screening and molecular modeling studies

A new series of benzimidazothiazole derivatives has been synthesized. The structure of the products was confirmed by spectroscopic techniques such as IR, NMR and mass spectroscopy. The tested compounds were evaluated for their anti-inflammatory activity either in vitro through the COX enzyme inhibition assay, or in vivo through carrageenan paw edema technique. Results revealed that compound 25 and 29 represented the most active ones among the entire series with % inhibition 72.19, 72.07 for COX-1, and 87.46, 87.38 for COX-2, respectively. Interestingly, all synthesized compounds exhibited IC50 values less than both reference drugs celecoxib and naproxen, indicating their superior potency. For compound 25, it showed about 340 and 198 times more potent than celecoxib and naproxen respectively as COX-1 inhibitor (IC50 value 0.044 vs. 15.000 and 8.700 µM), and 10 and 115 times more potent than the same drugs as COX-2 inhibitor (IC50 value 4.52 vs. 40.00 and 520.00 nM). The antitumor activity of the products was also evaluated and the results obtained are consistent with those obtained by the anti-inflammatory screening where compounds 25 and 29 proved to be the most active ones among the other compounds with %GI ranging from 31.5 to 62.5% and they exhibited the lowest IC50 values as well. The ADMET analysis of the tested compounds was also performed in addition to the molecular modeling studies that included flexible alignment, surface and electrostatic maps in addition to the Lipinisk's rule of five.

Synthesis of New Triarylpyrazole Derivatives Possessing Terminal Sulfonamide Moiety and Their Inhibitory Effects on PGE₂ and Nitric Oxide Productions in Lipopolysaccharide-Induced RAW 264.7 Macrophages.

This article describes the design, synthesis, and in vitro anti-inflammatory screening of new triarylpyrazole derivatives. A total of 34 new compounds were synthesized containing a terminal arylsulfonamide moiety and a different linker between the sulfonamide and pyridine ring at position 4 of the pyrazole ring. All the target compounds were tested for both cytotoxicity and nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Compounds 1b, 1d, 1g, 2a, and 2c showed the highest NO inhibition percentages and the lowest cytotoxic effect. The most potent derivatives were tested for their ability to inhibit prostaglandin E2 (PGE2) in LPS-induced RAW 264.7 macrophages. The IC50 for nitric oxide inhibition, PGE2 inhibition, and cell viability were determined. In addition, 1b, 1d, 1g, 2a, and 2c were tested for their inhibitory effect on LPS-induced inducible nitric oxide synthase (iNOS) and Cyclooxygenase 2 (COX-2) protein expression as well as iNOS enzymatic activity.

Antitubercular and anti-inflammatory properties screening of natural products from Plectranthus species.

Confirm the use ofPlectanthus spp. plants in traditional medicine, particularly as anti-inflammatory and anti-infective agents. Compounds previously isolated fromPlectranthusspp. were studied for their anti-inflammatory activity using the SNAP assay and RAW 264.7 cells, by the quantification of nitric oxide. An halimane diterpene and its derivatives were tested in infected macrophages withM. tuberculosisH37Rv, using CFU counts assay, at their minimum inhibitory concentration values.Results: The isolated compounds tested at noncytotoxic concentrations, did not reveal nitric oxide scavenging in the S-nitroso-N-acetylpenicillamine and the cellular assays. On the other hand, promising results were obtained regarding one semisynthetic halimane derivative (11R*,13E)-halima-5,13-diene-11,15-diol), previously prepared (2.1×105CFU/mL), with an effect similar to the antitubercular drugs ethambutol (2.0×105CFU/mL) and isoniazid (1.2×105CFU/mL). The present report demonstrates the relevance of Plectranthus spp. in medicinal chemistry drug development for TB and other infective respiratory complaints. Also, this work suggests that further studies involving other inflammatory mediators are needed to validate the anti-inflammatory use of these medicinal plants.

Design, synthesis and evaluation of some pyrazolo[3,4-d]pyrimidine derivatives bearing thiazolidinone moiety as anti-inflammatory agents

Two new series of pyrazolo[3,4-d]pyrimidine bearing thiazolidinone moiety were designed and synthesized. The newly synthesized compounds were evaluated for their in vitro (COX-1 and COX-2) inhibitory assay. Compounds that showed promising COX-2 selectivity were further subjected to in vivo anti-inflammatory screening applying formalin induced paw edema (acute model) and cotton-pellet induced granuloma (chronic model) assays using celecoxib and diclofenac sodium as reference drugs. The histopathological and ulcerogenic potential were also determined. In vivo anti-inflammatory data showed that compounds 2, 6, 7d displayed anti-inflammatory activity higher than both references in the formalin induced paw edema model. On the other hand, compounds 2, 3d, 3e, 7b and 7d displayed anti-inflammatory activity greater than or nearly equivalent to diclofenac sodium in the cotton pellet-induced granuloma assay. Moreover, most of the tested compounds revealed good gastrointestinal safety profile. Collectively, compounds 2 and 7d were considered as promising candidates in managing both acute and chronic inflammation with safe gastrointestinal margin.

Engineering a yeast double-molecule carrier for drug screening

With the advantages of unicellular eukaryotic structure and easy manipulation, yeast becomes a popular tool for biochemical, genetic and medicinal studies. In order to construct an efficient anti-inflammatory drug screening platform, we engineered yeast as a double-molecule carrier, of which an inserted domain (I domain) of lymphocyte function-associated antigen 1 was displayed on yeast surface and a green fluorescent protein (GFP) was expressed inside cytosol. The I domain specifically targeted a surface marker of mammalian cells, intercellular adhesion molecule 1, whose number is correlated with the level of cellular inflammation. Examination of GFP intensity enables swift quantification of the yeast-mammalian cell binding and thus it reflects inflammatory potency, herein the inflammatory index, of a chemical imposed to cells. The inflammatory potency of a total of 1340 chemicals was indexed. Among them, 1 inflammation-inducing and 1 inflammation-reducing compounds were verified both in vitro and in vivo. Our method demonstrated a swift, facile and high-throughput screening platform at the protein level for inflammation and related diseases drug discovery without using sophisticated instruments.

Synthesis, anti-inflammatory screening, molecular docking, and COX-1,2/-5-LOX inhibition profile of some novel quinoline derivatives

New quinoline compounds comprising pyrazole scaffold through different amide linkages were synthesized. The synthesized compounds were evaluated for their anti-inflammatory activity. Eight compounds (5c, 11b,c, 12c, 14a,b, 20a and 21a) were found to exhibit promising anti-inflammatory profiles in acute and sub-acute inflammatory models. They were screened for their ulcerogenic activity and none of them showed significant ulcerogenic activity comparable to the reference drug celecoxib and are well tolerated by experimental animals with high safety margin (ALD50 > 0.3 g/kg). Compounds 5c, 11b,c, 12c, 14a,b, 20a and 21a showed significant in vitro LOX inhibitory activity higher than that of zileuton. In vitro COX-1/COX-2 inhibition study revealed that compounds 12c, 14a,b and 20a showed higher selectivity towards COX-2 than COX-1. Among the tested compounds, 12c, 14a and 14b showed the highest inhibitory activity against COX-2 with an IC50 values of 0.1, 0.11 and 0.11 μM respectively. The docking experiments attempted to postulate the binding mode for the most active compounds in the binding site of COX-2 enzymes and confirmed the high selectivity binding towards COX-2 enzyme over COX-1.

Microfluidic Coculture Device for Monitoring of Inflammation-Induced Myocardial Injury Dynamics.

Emerging awareness of cardiac macrophages' role in inflammation after myocardial infarction indicates that overabundant proinflammatory macrophages induce accentuated myocardial injury. The investigation of the macrophages-cardiomyocytes interaction and inflammation-induced dynamic damage in myocardial infarction, especially in a spatiotemporally controlled manner, remains a huge challenge. Here, we developed an in vitro model using a microfluidic coculture system to mimic inflammatory cardiac injury. To our knowledge, on-chip pathological models focused on inflammation-induced myocardial injury have not been reported. The device consists of two sets of thin interconnecting grooves that isolate heterogeneous cells spatially but maintain their soluble factors communication. The mass transportation is visually characterized, and the complete diffusion reaches equilibrium within 100 s. We investigate the dynamic interaction between the macrophages and the cardiomyocytes in the spatiotemporal controlled microenvironment, mimicking a key aspect of the in vivo pathophysiological process. The results show that the activated macrophages induce time-lapsed apoptotic responses of the cardiac cells and damage mitochondria membrane integrity. The anti-inflammatory and cardio-protective effects of quercetin were explored on the chip. The extent of caspase-3 activation is asynchronous in the individual cardiac cells, suggesting the different apoptosis dynamics. We further demonstrate that the mechanism of activated inflammation is associated with the upregulation of several inflammatory cytokines and NF-κB pathway. Thus, the developed microfluidic coculture device provides a useful tool for real-time monitoring of inflammatory response for myocardial disease and holds potential for anti-inflammatory drug screening.

A novel triterpenoid 16-hydroxy betulinic acid isolated from Mikania cordata attributes multi-faced pharmacological activities.

The aerial parts of extensively used ethnomedicinal plant Mikania cordata (Burm. f.) Robinson growing wild in Bangladesh were investigated to isolate and characterize compounds responsible for the bioactivities of the plant. In the present study, a new derivatives of betulinic acid, 16-hydroxy betulinic acid [3β,16-dihydroxy-lup-20(29)-en-28-oic] was isolated and the structure of the compound was determined by NMR spectroscopic means and comparing with available literature data. The isolated compound was then investigated for different pharmacological activities including antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic potential employing different methods. The compound showed potent antibacterial activity with inhibition zone of diameter ranging from 12.0 to 17.5 mm and antifungal activity with mycelial growth inhibition ranging from 37.6 to 54.5%. The MIC values for antibacterial and antifungal activities ranged from 31.5–125 and 250–1000 μg/mL respectively. The compound (50 and 100 mg/kg body weight) showed potent peripheral and central analgesic activity with 55.19% and 41% of writhing inhibition at 90 min after administration of the compound and the highest 55.98%, 79.18% elongation of reaction time, respectively. In anti-inflammatory activity screening, the compound (100 mg/kg b.w.) revealed the highest 77.08% edema inhibition at 4 h after administration of carrageenan. In antipyretic assay, 16-hydroxy betulinic acid displayed a strong antipyretic effect in yeast-induced rats. From the present study it is apparent that 16-hydroxy betulinic acid might play vital role to establish M. cordata as ethnomedicinal plant to treat wound, cuts and fever.

Evaluation of anti-nociceptive, anti-inflammatory and antipyretic potential of Mikania cordata (Burm. f.) Robinson in experimental animal model

Mikania cordata is widely used for the treatment of cuts, wounds, and dengue fever in Bangladesh. In the present study, essential oil (12.5, 25 and 50 mg/kg) and two extracts, viz., chloroform and ethyl acetate extracts (200, 400, 800 mg/kg b.w.) were tested for peripheral and central anti-nociceptive activity by acetic acid-induced writhing and hot plate method, respectively. Carrageenan-induced rat paw edema assay and yeast-induced hyperthermia assay were also carried out to evaluate anti-inflammatory and antipyretic properties of oil and extracts, respectively at aforesaid doses. The essential oil (50 mg/kg), chloroform extract (800 mg/kg) and ethyl acetate extract (800 mg/kg) showed potent peripheral anti-nociceptive activity having 47.33%, 29.33% and 16.65% of writhing inhibition, respectively, comparable with standard diclofenac (52.0%). Essential oil (50 mg/kg), chloroform extract (800 mg/kg) and ethyl acetate extract (800 mg/kg) presented promising central anti-nociceptive activity as well having 95.86%, 79.18% and 42.37% elongation of reaction time, respectively, at 90 min after administration of essential oil, ethyl acetate extract and 60 min after administration of chloroform extract. In anti-inflammatory activity screening, the essential oil (50 mg/kg) produced the highest 72.80% edema inhibition at 4 h after administration of carrageenan which was comparable with that of standard phenylbutazoe (87.87%). On the other hand, chloroform extract (800 mg/kg) and ethyl acetate extract (800 mg/kg) showed up to 34.31% and 15.27% of edema inhibition, respectively, at 4 h after administration of carrageenan. In antipyretic assay, the essential oil and chloroform extract displayed a strong antipyretic effect in yeast-induced rats, whereas the ethyl acetate extract had no antipyretic activity. The present study revealed anti-nociceptive, anti-inflammatory and antipyretic potential of M. cordata which could be the therapeutic option against fever, inflammations as well as painful conditions and confirmed the traditional use of M. cordata.

Synthesis, biological evaluation and molecular modeling of novel thienopyrimidinone and triazolothienopyrimidinone derivatives as dual anti-inflammatory antimicrobial agents

New thienopyrimidinone and triazolothienopyrimidinone derivatives have been synthesized. These compounds were subjected to anti-inflammatory and antimicrobial activity screening aiming to identify new candidates that have dual anti-inflammatory and antimicrobial activities.Compounds 5, 7 and 10a showed minimal ulcerogenic effect and high selectivity towards human recombinant COX-2 over COX-1 enzyme. Their docking outcome correlated with their biological activity and assured the high selectivity binding towards COX-2. In addition, they could act safely up to 80 mg/kg orally or 40 mg/kg parentrally. The antimicrobial screening showed that compound 10a displayed distinctive inhibitory effect on the growth of Escherichia coli comparable to that of ampicillin. Moreover, compounds 5, 7, 9 and 12a possessed 50% of the inhibitory activity of ampicillin against E. coli. Thus, compounds 5, 7 and 10a represent promising dual acting anti-inflammatory and antimicrobial agents. This work provides rewarding template enriching the chemical space for dual anti-inflammatory anti-microbial activities.

ANTI-INFLAMMATORY ACTIVITY OF BACTERIA ASSOCIATED WITH MARINE SPONGE (HALICLONA AMBOINENSIS) VIA REDUCTING NO PRODUCTION AND INHIBITING CYCLOOXYGENASE-1, CYCLOOXYGENASE-2, AND SECRETORY PHOSPHOLIPASE A2 ACTIVITIES

Objective: This study aimed to investigate the anti-inflammatory activity of methanol extract and fractions of bacteria associated with sponge (Haliclona amboinensis) and to evaluate their effect in reducing NO production and inhibiting cyclooxygenase-1 (COX-1), cyclooxgenase-2 (COX-2) and secretory phospholipase A2 (sPLA2) activity.Methods: All bacterial isolates were cultured and supernatants were collected for the extraction of secondary metabolites using diaion HP-20 to obtain methanol extracts. Evaluation of cytotoxicity property was carried out on macrophage cell lines (RAW264.7) by 3-(4,5-dimethylthiazol- 2-yl) 2,5-diphenyl tetrazoliumbromide assay. Anti-inflammatory screening was done by inducible nitric oxide assay on RAW264.7 cell lines with lipopolysaccharide (LPS) stimulation. Dianion HP-20 was used to remove salt content. A selected methanol extract was subjected to further fractionations by C-18 reverse phase and their anti-inflammatory potential was evaluated by COX-1 and COX-2, and sPLA2 enzymatic assay.Results: Seven methanol extracts showed no cytotoxic property against RAW 264.7 cell line (inhibitory concentration 50% > 30 μg/ml) and selected for anti-inflammatory screening assay. Result showed methanol extract HM 1.2 reduced NO production >80% and it has been selected for phytochemical screening, further fractionations and assay. Phytochemical screening showed alkaloids and terpenoids present in the HM 1.2. The HM 1.2 and its fractions (F1, F2, F1C1, F1C2, F1C3, and F1C4) were proven to inhibit COX-1, COX-2, and sPLA2 activity in the range of 60.516-116.886%, 20.554- 116.457%, and 70.2667-114.8148%, respectively.Conclusions: This study revealed that bacteria associated with H. amboinensis have produced anti-inflammatory activity via reducing NO production and inhibiting COX-1, COX-2, and sPLA2 activity.

COX Inhibition Profile and Molecular Docking Studies of Some 2-(Trimethoxyphenyl)-Thiazoles.

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used therapeutic agents that exhibit frequent and sometimes severe adverse effects, including gastrointestinal ulcerations and cardiovascular disorders. In an effort to obtain safer NSAIDs, we assessed the direct cyclooxygenase (COX) inhibition activity and we investigated the potential COX binding mode of some previously reported 2-(trimethoxyphenyl)-thiazoles. The in vitro COX inhibition assays were performed against ovine COX-1 and human recombinant COX-2. Molecular docking studies were performed to explain the possible interactions between the inhibitors and both COX isoforms binding pockets. Four of the tested compounds proved to be good inhibitors of both COX isoforms, but only compound A3 showed a good COX-2 selectivity index, similar to meloxicam. The plausible binding mode of compound A3 revealed hydrogen bond interactions with binding site key residues including Arg120, Tyr355, Ser530, Met522 and Trp387, whereas hydrophobic contacts were detected with Leu352, Val349, Leu359, Phe518, Gly526, and Ala527. Computationally predicted pharmacokinetic profile revealed A3 as lead candidate. The present data prove that the investigated compounds inhibit COX and thus confirm the previously reported in vivo anti-inflammatory screening results suggesting that A3 is a suitable candidate for further development as a NSAID.

Some Novel Mannich Bases of 5-(3,4-Dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one and Their Anti-Inflammatory Activity.

Non-steroidal anti-inflammatory drugs (NSAIDs), which are widely used for the treatment of rheumatic arthritis, pain, and many different types of inflammatory disorders, cause serious gastrointestinal (GI) side effects. The free carboxylic acid group existing on their chemical structure is correlated with GI toxicity related with all routine NSAIDs. Replacing this functional group with the 1,3,4-oxadiazole bioisostere is a generally used strategy to obtain an anti-inflammatory agent devoid of GI side effects. In the present work, a novel group of 5-(3,4-dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one Mannich bases were synthesized and characterized on the basis of IR, 1 H NMR, and elemental analysis results. The target compounds were first tested for cytotoxicity to determine a non-toxic concentration for anti-inflammatory screening. Anti-inflammatory effects of the compounds were evaluated by in vitro lipopolysaccharide (LPS)-induced NO production and in vivo carrageenan footpad edema with ulcerogenic profile. In LPS-induced RAW 264.7 macrophages, most of the compounds showed inhibitory activity on nitrite production while compounds 5a, 5h, and 5j exhibited the best profiles by suppressing the NO production. To evaluate the in vivo anti-inflammatory potency of the compounds, the inflammatory response was quantified by increment in paw size in the carrageenan footpad edema assay. The anti-inflammatory data scoring showed that compounds 5a-d, 5g, and 5j, at the dose of 100 mg/kg, exhibited anti-inflammatory activity, which for compound 5g was comparable to that of the reference drug indomethacin with 53.9% and 55.5% inhibition in 60 and 120 min, respectively.

Identification and application of anti-inflammatory compounds screening system based on RAW264.7 cells stably expressing NF-\u03baB-dependent SEAP reporter gene

BackgroundNF-κB is one of the key transcription factors in the inflammatory response, transactivates a series of pro-inflammatory genes and is therefore regarded as an important target for anti-inflammatory drug screening.MethodWe recombined the reporter gene vector with inserting the “neo” transcript into the vector pNF-κB-SEAP, made the reporter gene vector stable in a eukaryotic cell line. The recombinant reporter gene vector we named pNF-κB-SEAP-Neo was transfected into RAW264.7. We selected the transfected RAW264.7 cell line with G418 for 15 days and then get RAW264.7 cells stably expressing NF-κB-dependent SEAP named as RAW264.7-pNF-κB-SEAP cells. We treated the RAW264.7-pNF-κB-SEAP cells with NF-κB agonists as LPS, PolyI:C and TNF-α, NF-κB inhibitor as PDTC and BAY117085, in different concentrations and time points and tested the expression of the SEAP, constructed the drug screening system on the base of the RAW264.7-pNF-κB-SEAP cell line. 130 chemicals were screened with the drug screening system we constructed and one of these chemicals numbered w10 was found could inhibit the NF-κB significantly. At last, we verified the inhibition of w10 to expression of genes promoted with NF-κB in HepG2 and Hela, and to migration of Hela.ResultIn this study, we established a drug screening system based on RAW264.7 cells that stably expressed the NF-κB-dependent, SEAP reporter gene. To develop a standard method for drug screening using this reporter-gene cell line, the test approach of SEAP was optimized and basic conditions for drug screening were chosen. This included the initial cell number inoculated in a 96-well plate, the optimum agonist, inhibitor of NF-κB pathway and their concentrations during screening. Subsequently, 130 newly synthesized compounds were screened using the stable reporter-gene cell line. The anti-inflammatory effects of the candidate compounds obtained were further verified in 2 cancer cell lines. The results indicated that compound W10 (methyl 4-(4-(prop-2-yn-1-ylcarbamoyl) phenylcarbamoyl) benzoate) significantly inhibited SEAP production under the screening conditions. Further results confirmed that the precursor compound significantly inhibited the transcription of NF-κB target genes.ConclusionIn conclusion, RAW264.7 cells, stably expressing the NF-κB-dependent SEAP-reporter gene, may provide a new, feasible, and efficient cellular drug-screening system.

Design, synthesis and pharmacological analysis of 5-[4'-(substituted-methyl)[1,1'-biphenyl]-2-yl]-1H-tetrazoles.

In the present paper 5-[4'-({4-[(4-aryloxy)methyl]-1H-1,2,3-triazol-1-yl}methyl)[1,1'-biphenyl]-2-yl]-1H-tetrazoles (5a-g) and [2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl-substituted-1-carbodithioates (11h-q) have been designed and synthesized. These compounds were subjected to docking (against AT1 receptor protein enzyme in complex with Lisinopril), in vitro angiotensin converting enzyme inhibition, anti-proliferative, anti-inflammatory screening (through egg albumin denaturation inhibition and red blood cell membrane stabilization assay) and finally anti-fungal activity analyses. Some of the compounds have shown significant pharmacological properties.

Screening of Luzula species native to the Carpathian Basin for anti-inflammatory activity and bioactivity-guided isolation of compounds from Luzula luzuloides (Lam.) Dandy & Wilmott

The present study focused on the anti-inflammatory screening of Luzula species native to the Carpathian Basin and bioactivity-guided isolation of compounds of Luzula luzuloides (Lam.) Dandy & Wilmott. The anti-inflammatory properties of extracts with different polarity prepared from Luzula species were determined. Among them, the CH2Cl2-soluble fraction of L. luzuloides possessed strong inhibitory effects on superoxide anion generation (99.39±0.37%) and elastase release (114.22±3.13%) in fMLP/CB-induced human neutrophils at concentration of 10μg/mL. From this fraction, six compounds (1–6) were isolated by the combination of different chromatographic methods. The structures of the compounds were determined by means of MS, 1D and 2D NMR spectroscopy. The results allowed the identification of the new 1,6-dihydroxy-2-keto-1,7-dimethyl-8-vinyl-1,2-dihydrophenanthrene (1) from the plant, named luzulin A. Chiral HPLC and HPLC-ECD analysis revealed that 1 possesses low enantiomeric excess and TDDFT-ECD calculations afforded the configurational assignment of the separated enantiomers. Three known phenanthrenes [juncuenin B (2), dehydrojuncuenin B (3) and juncusol (4)] and two flavonoids [apigenin (5) and luteolin (6)] were also isolated. The anti-inflammatory activity of the isolated compounds was tested and IC50 values were determined. This was the first time that phenanthrenes were detected in a Luzula species. The oxidative transformation of juncuenin B (3) led to the isolation of its possible biometabolites, namely luzulin A (1), dehydrojuncuenin B (4), and juncuenin D (7). The isolated compounds (1–4) confirm that besides flavonoids, phenanthrenes could also serve as chemotaxonomic markers for Luzula species and prove the close relationship of Juncus and Luzula genus.

Anti-inflammatory screening of ethanolic leaf extract of Vernonia arborea Buch. –Ham.in formalin induced albino wistar rats

Anti-inflammatory screening of ethanolic leaf extract of Vernonia arborea Buch. –Ham.in formalin induced albino wistar rats

Synthesis of New Tricyclic and Tetracyclic Fused Coumarin Sulfonate Derivatives and Their Inhibitory Effects on LPS-Induced Nitric Oxide and PGE2 Productions in RAW 264.7 Macrophages: Part 2.

The synthesis of a new series of 21 fused coumarin derivatives is described, and the biological evaluation of their in vitro antiinflammatory effects as inhibitors of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2 ) production in RAW 264.7 macrophages. The target compounds 1a-u were first tested for cytotoxicity to determine a non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE2 production would not be caused by cytotoxicity. Compounds 1f and 1p were the most active PGE2 inhibitors with IC50 values of 0.89 and 0.95 µM, respectively. Western blot and cell-free COX-2 screening showed that their effects were due to inhibition of both COX-2 protein expression and COX-2 enzyme activity. Their IC50 values against the COX-2 enzyme were 0.67 and 0.85 µM, respectively, which is more potent than etoricoxib. The selectivity indexes of compounds 1f and 1p against COX-2 compared to COX-1 were 41.1 and 42.5, respectively. Compound 1f showed strong inhibitory effects at 5 µM concentration on COX-2 mRNA expression in LPS-induced RAW 264.7 macrophages. Moreover, the tricyclic compounds 1l and 1n as well as the tetracyclic analog 1u were the most potent NO inhibitors, with one-digit micromolar IC50 values. They showed dose-dependent inhibition of inducible nitric oxide synthase (iNOS) protein expression. The tetracyclic derivative 1u was the most potent inhibitor of NO production. It also exhibited a strong inhibitory effect on iNOS mRNA expression in LPS-induced RAW 264.7 macrophages.

Anti-inflammatory active fraction screening and mechanism of unripe Forsythiae Fructus based on ¹H-NMR metabolomics

To screen the anti-inflammatory active fraction of unripe Forsythiae Fructus, and elucidate the action mechanism, water decoction, ethyl acetate portion, n-butanol portion and residue water extracts of unripe Forsythiae Fructus were administered into rats for continuously 15 days. The acute lung injury inflammatory model was established to observe the section structure of lung tissues. Levels of IL-6, TNF-α, IL-1β and IL-10 in bronchoalveolar lavage fluid were determined by ELISA kits, and changes in endogenous metabolites in serum were analyzed based on 1H-NMR metabolomics. The results showed that ethyl acetate portion of unripe Forsythiae Fructus had a better anti-inflammatory activity against acute lung injury, and could suppress the release of inflammatory factors of IL-6, TNF-α, IL-1β, significantly reduce contents of creatine, β-OH-butyrate, succinate, lysine, valine, isoleucine and glutamine, and elevate the content of GPC in serum. Ethyl acetate portion was proved to be the main fraction of anti-inflammatory activity from the perspective of endogenous metabolites in serum, and played an anti-inflammatory role by regulating creatine metabolism, choline metabolism, branched-chain amino acid metabolism and TCA cycles. This study could lay a foundation for studying pharmacodynamic material basis of unripe Forsythiae Fructus.