Isosibiricin inhibits microglial activation by targeting the dopamine D1/D2 receptor-dependent NLRP3/caspase-1 inflammasome pathway.

Microglia-mediated neuroinflammation is a crucial risk factor for neurological disorders. Recently, dopamine receptors have been found to be involved in multiple immunopathological processes and considered as valuable therapeutic targets for inflammation-associated neurologic diseases. In this study we investigated the anti-neuroinflammation effect of isosibiricin, a natural coumarin compound isolated from medicinal plant Murraya exotica. We showed that isosibiricin (10-50 µM) dose-dependently inhibited lipopolysaccharide (LPS)-induced BV-2 microglia activation, evidenced by the decreased expression of inflammatory mediators, including nitrite oxide (NO), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and interleukin-18 (IL-18). By using transcriptomics coupled with bioinformatics analysis, we revealed that isosibiricin treatment mainly affect dopamine receptor signalling pathway. We further demonstrated that isosibiricin upregulated the expression of dopamine D1/2 receptors in LPS-treated BV-2 cells, resulting in inhibitory effect on nucleotide binding domain-like receptor protein 3 (NLRP3)/caspase-1 inflammasome pathway. Treatment with dopamine D1/2 receptor antagonists SCH 23390 (1 µM) or sultopride (1 µM) could reverse the inhibitory effects of isosibiricin on NLRP3 expression as well as the cleavages of caspase-1 and IL-1ß. Collectively, this study demonstrates a promising therapeutic strategy for neuroinflammation by targeting dopamine D1/2 receptors.

Anti-inflammatory pterocarpanoids from the roots of Pongamia pinnata.

A phytochemical study on the roots of Pongamia pinnata afforded 11 pterocarpanoids, including three new compounds. The structures of the isolated compounds were determined by 1D and 2D NMR and HRESIMS data. The absolute configurations of the new compounds were assigned via analysis of the specific rotations and electronic circular dichroism (ECD) spectra. The isolates were evaluated for their inhibitory effects on nitric oxide (NO) production in LPS-stimulated BV-2 microglial cells. Six compounds exhibited inhibitory effects against NO production, and compound 5 showed the best activity with an IC50 value at 12.0 µM.

Anti-inflammatory flavone and chalcone derivatives from the roots of Pongamia pinnata (L.) Pierre.

A phytochemical study on the roots of Pongamia pinnata (L.) Pierre yielded 52 flavonoids, including four previously undescribed flavone and four previously undescribed chalcone derivatives. The structures of the isolated compounds were determined on the basis of the 1D, 2D NMR, and mass spectroscopic data. The absolute configurations of the compounds were assigned via the specific rotation, Mosher's method, as well as the electronic circular dichroism (ECD) spectra. All the isolates were evaluated for their inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells. Ten compounds showed significant inhibitory effects against NO production, comparable to the positive control, dexamethasone.

Pyrrolo[2,1-a]isoquinoline and pyrrole alkaloids from Sinomenium acutum.

Two pyrrolo[2,1-a]isoquinolines (1 and 2) and three pyrrole alkaloids (3-5), including three new ones, named sinopyrines A-C (1-3), were isolated from the 95% EtOH extract of the stems and rhizomes of Sinomenium acutum (Thumb.) Rehd. et Wils. The structures of the new compounds were elucidated on the basis of spectroscopic data. This is the first report of pyrrole-bearing natural compounds from the family Menispermaceae.

A novel and practical synthesis of CAT3: a phenanthroindolizidine alkaloid with potential in treating glioblastoma.

CAT3, one of the (+)-deoxytylophorinine-based phenanthroindolizidine alkaloids, is a promising therapeutic agent for the treatment of hedgehog (Hh)-driven glioblastoma and is currently being evaluated in preclinical studies. In this paper, a novel and practical synthetic route for CAT3 was firstly demonstrated with 10% overall yield in 11 steps and has been successfully validated for pilot-plant scale preparation. Investigation of the substitution at the 3-position of phenanthrene revealed that the electron-donating functionality can well preserve the S configuration. In particular, the excellent enantiomeric excess of CAT3 (≥99% ee) was achieved by introducing the strongly electron-donating tert-butyldimethylsilyl (TBS) group.

Highly Selective Activation of Heat Shock Protein 70 by Allosteric Regulation Provides an Insight into Efficient Neuroinflammation Inhibition.

Heat shock protein 70 (Hsp70) is widely involved in immune disorders, making it as an attractive drug target for inflammation diseases. Nonselective induction of Hsp70 upregulation for inflammation therapy could cause extensive interference in inflammation-unrelated protein functions, potentially resulting in side effects. Nevertheless, direct pharmacological activation of Hsp70 via targeting specific functional amino acid residue may provide an insight into precise Hsp70 function regulation and a more satisfactory treatment effect for inflammation, which has not been extensively focused. Here we show a cysteine residue (Cys306) for selective Hsp70 activation using natural small-molecule handelin. Covalent modification of Cys306 significantly elevates Hsp70 activity and shows more satisfactory anti-neuroinflammation effects. Mechanism study reveals Cys306 modification by handelin induces an allosteric regulation to facilitate adenosine triphosphate hydrolysis capacity of Hsp70, which leads to the effective blockage of subsequent inflammation signaling pathway. Collectively, our study offers some insights into direct pharmacological activation of Hsp70 by specially targeting functional cysteine residue, thus providing a powerful tool for accurately modulating neuroinflammation pathogenesis in human with fewer undesirable adverse effects.

Highly selective inhibition of IMPDH2 provides the basis of antineuroinflammation therapy.

Inosine monophosphate dehydrogenase (IMPDH) of human is an attractive target for immunosuppressive agents. Currently, small-molecule inhibitors do not show good selectivity for different IMPDH isoforms (IMPDH1 and IMPDH2), resulting in some adverse effects, which limit their use. Herein, we used a small-molecule probe specifically targeting IMPDH2 and identified Cysteine residue 140 (Cys140) as a selective druggable site. On covalently binding to Cys140, the probe exerts an allosteric regulation to block the catalytic pocket of IMPDH2 and further induces IMPDH2 inactivation, leading to an effective suppression of neuroinflammatory responses. However, the probe does not covalently bind to IMPDH1. Taken together, our study shows Cys140 as a druggable site for selectively inhibiting IMPDH2, which provides great potential for development of therapy agents for autoimmune and neuroinflammatory diseases with less unfavorable tolerability profile.

Phloroglucinols with Antioxidant Activities Isolated from Lysidice rhodostegia.

Two new phloroglucinols, lysidisides X and Y (1 and 2), and two known compounds, 2-(2-methylbutyryl)phloroglucinol 1-O-ß-d-glucopyranoside (3) and (E)-resveratrol 3-(6″-galloyl)-O-ß-d-glucopyranoside (4), have been isolated from the roots of Lysidice rhodostegia. The structures of 1 and 2 were elucidated primarily by NMR experiments. Their absolute configurations were deduced via circular dichroism (CD) data and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 exhibited significant antioxidative activities with IC50 values of 12.0 and 11.8 µM, respectively.

Rhodomollins A and B, two Diterpenoids with an Unprecedented Backbone from the Fruits of Rhododendron molle.

Two new grayanoids, rhodomollin A (1) and rhodomollin B (2), possessing an unprecedented D-homo grayanane carbon skeleton, were isolated from the fruits of Rhododendron molle. The structures of 1 and 2 were fully characterized using a combination of spectroscopic analyses and X-ray crystallography. Rhodomollin B (2) exhibited modest activity against influenza virus A/95-359, with an IC50 value of 19.24 µM.

Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata.

Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia, and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pain, such as headache, rheumatic pain, toothache, and snake bites. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor transient receptor potential vanilloid 1 (TRPV1) channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from the active extract of Murraya alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1-expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice but not in TRPV1 knock-out mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy.

Antinociceptive Grayanoids from the Roots of Rhododendron molle.

Nine new grayanoids (1-9), together with 11 known compounds, were isolated from the roots of Rhododendron molle. The structures of the new compounds (1-9) were determined on the basis of spectroscopic analysis, including HRESIMS, and 1D and 2D NMR data. Compounds 4, 6, 12, and 14-20 showed significant antinociceptive activities in an acetic acid-induced writhing test. In particular, 14 and 15 were found to be more potent than morphine for both acute and inflammatory pain models and 100-fold more potent than gabapentin in a diabetic neuropathic pain model.

Biological Activity and Chemical Constituents of Essential Oil and Extracts of Murraya microphylla.

Murraya microphylla is the most closely related species to M. koenigii (Curry tree). Inspired by the traditional effects of M koenigii, the antioxidant, anti- inflammatory, and cytotoxic activities of the essential oil and extracts of M. microphylla were evaluated for the first time. The light petroleum and chloroform extracts were found to be able to scavenge DPPH free radicals, inhibit linoleic acid peroxidation, and nitric oxide production, as well as to present cytotoxicity to the human cancer cell lines HepG2, Bel7402, Bel7403, and Hela, but the essential oil only showed moderate activities. Chemical analysis of the active extracts by LC-DAD-MS" indicated that carbazole alkaloids were the main constituents. GC-MS analysis of the essential oil resulted in identification of 91 constituents, representing 96.9% of the total oil, with (E)-caryophyllene (18.4%) and terpinen-4-ol (12.6%) as the major constituents. These results demonstrate that M microphylla has similar biological activities, as well as chemical constituents to M. koenigii, and the carbazole alkaloids were disclosed to be the main potential active components. A promising development as a flavor and potential therapeutic agent could thus be predicated for this plant.

Caruifolin D from artemisia absinthium L. inhibits neuroinflammation via reactive oxygen species-dependent c-jun N-terminal kinase and protein kinase c/NF-κB signaling pathways.

This work aims to evaluate the anti-neuroinflammatory effects of natural sesquiterpene dimer caruifolin D from Artemisia absinthium L., which is an edible vegetable or traditional medicinal food in East Asia due to its sedation, anti-asthma and antipruritic effects. In this study, we reported that caruifolin D significantly inhibited the productions of various neuroinflammatory mediators from microglia in response to bacterial lipopolysaccharide stimulation. Moreover, anti-inflammatory mechanism study showed that caruifolin D markedly suppressed the production of intracellular reactive oxygen species, which was an important player involved in neuroinflammation, leading to inhibitory effects on the activations of protein kinase C (PKC) and c-Jun N-terminal kinase (JNK), which were two major neuroinflammatory signaling pathways in the brains. Furthermore, caruifolin D protected neurons against microglia-mediated neuronal inflammatory damages by up-regulating neuronal viability and maintaining healthy neuronal morphology. Taken together, these results expanded our knowledge about the anti-neuroinflammatory and neuroprotective mechanism of Artemisia absinthium L., and also suggested that caruifolin D was a major anti-inflammatory component from Artemisia absinthium L., which might be developed as a drug candidate for neuroinflammation-related diseases.

Natural small molecule FMHM inhibits lipopolysaccharide-induced inflammatory response by promoting TRAF6 degradation via K48-linked polyubiquitination.

TNF receptor-associated factor 6 (TRAF6) is a key hub protein involved in Toll-like receptor-dependent inflammatory signaling pathway, and it recruits additional proteins to form multiprotein complexes capable of activating downstream NF-κB inflammatory signaling pathway. Ubiquitin-proteasome system (UPS) plays a crucial role in various protein degradations, such as TRAF6, leading to inhibitory effects on inflammatory response and immunologic function. However, whether ubiquitination-dependent TRAF6 degradation can be used as a novel anti-inflammatory drug target still remains to be explored. FMHM, a bioactive natural small molecule compound extracted from Chinese herbal medicine Radix Polygalae, suppressed acute inflammatory response by targeting ubiquitin protein and inducing UPS-dependent TRAF6 degradation mechanism. It was found that FMHM targeted ubiquitin protein via Lys48 site directly induced Lys48 residue-linked polyubiquitination. This promoted Lys48 residue-linked polyubiquitin chain formation on TRAF6, resulting in increased TRAF6 degradation via UPS and inactivation of downstream NF-κB inflammatory pathway. Consequently, FMHM down-regulated inflammatory mediator levels in circulation, protected multiple organs against inflammatory injury in vivo, and prolong the survival of endotoxemia mouse models. Therefore, FMHM can serve as a novel lead compound for the development of TRAF6 scavenging agent via ubiquitination-dependent mode, which represents a promising strategy for treating inflammatory diseases.

[Study on secondary metabolites of endophytic fungi Penicillium dangeardii].

Endophytic fungi Penicillium dangeardii, isolated from Lysidice rhodostegia Hance root, was fermented and the secondary metabolites were studied. By means of Sephadex LH-20 column chromatography, ODS column chromatography and PHPLC over the fermented culture, 5 compounds were isolated. By using ESI-MS and NMR, the structures of the compounds were determined as N-[9-(ß- D-ribofuranosyl)-9H-purin-6-yl]-L-aspartic acid (1), 3-caffeoylquinic acid (2), 4-caffeoylquinic acid (3), and 5-caffeoylquinic acid (4), 3-hydroxy-benzoic acid-4-O-ß-D-glucopyranoside (5).

Nitrogen Oxide Inhibitory Trimeric and Dimeric Carbazole Alkaloids from Murraya tetramera.

Two new structurally unique trimeric carbazole alkaloids, murratrines A and B (1, 2), and 11 new carbazole dimers, murradines A-K (3-13), and four known analogues (14-17) were isolated from the leaves and stems of Murraya tetramera. The structures and relative configurations of 1-13 were elucidated on the basis of comprehensive 1D and 2D NMR spectroscopy, high-resolution mass spectrometry, and electronic circular dichroism (ECD) data analysis. Murratrines A and B (1, 2) both contain an unprecedented carbazole trimeric skeleton, and murradines A and D (3, 6) are the first natural C-1-C-3'-methyl-linked and C-6-C-3'-methyl-linked dimeric carbazole alkaloids, respectively. Compounds 4, 10, 14, 15, and 17 exhibited inhibition of nitric oxide production stimulated by lipopolysaccharide in BV-2 microglial cells with IC50 values in the range of 11.2-19.3 µM.

[Simultaneous determination of benzophenones and xanthone in leaves of Aquilaria sinensis by RP-HPLC-UV].

This study is to develop a sensitive method by using reversed-phase high performance liquid chromatography coupled with UV detector (HPLC-UV) to simultaneously determine four bioactive compounds, iriflophenone 3-C-beta-D-glucoside, iriflophenone 3,5-C-beta-D-diglucoside, mangiferin, and iriflophenone 2-O-alpha-L-rhamnoside in the leaves of Aquilaria sinensis. An Agilent Zorbax SB-C, column (4, 6 mm x 250 mm, 5 microm) was used, and the gradient elution was performed with mobile phase of 0.1% aqueous phosphoric acid and acetonitrile at a flow rate of 1 mL x min(-1). The detection wavelength was 280 nm, and the column temperature was 25 degrees C. The four marker compounds were well separated with good linearity (R2 > 0.9990), precision, stability and repeatabili y. The-recovery rates were in the range of 98.80%-101.39%. For 15 branch of the leaves, the contents of iriflophenone 3-C-beta-D-gluoside, iriflophenone 3,5-C-beta-D-diglucoside, mangiferin, and iriflophenone 2-O-alpha-L-rhamnoside were between 0.41-14.48, 0.72-3.85, 4.30-29.07, 0.24-5.06 mg, respectivley. This method is precise, accurate and reliable, which provides an efficient way for the quality control of the leaves of A. sinensis. This will promote the comprehensive usage of this plant.

Anti-Neuroinflammatory Effect of MC13, a Novel Coumarin Compound From Condiment Murraya, Through Inhibiting Lipopolysaccharide-Induced TRAF6-TAK1-NF-κB, P38/ERK MAPKS and Jak2-Stat1/Stat3 Pathways.

MC13 is a novel coumarin compound found in Murraya, an economic crop whose leaves are widely used as condiment (curry) in cuisine. The aims of the present study were to investigate the neuroprotective effects of MC13 on microglia-mediated inflammatory injury model as well as potential molecular mechanism. Cell viability and apoptosis assay demonstrated that MC13 was not toxic to neurons and significantly protected neurons from microglia-mediated inflammatory injury upon lipopolysaccharide (LPS) stimulation. Results showed that MC13 markedly inhibited LPS-induced production of various inflammatory mediators, including nitrite oxide (Griess method), TNF-α and IL-6 (ELISA assay) in a concentration-dependent manner. Mechanism study showed that MC13 could suppress the activation of NF-κB, which was the central regulator for inflammatory response, and also decreased the interaction of TGF-ß-activated kinase 1 (TAK1)-binding protein (TAB2) with TAK1 and TNF receptor associated factor (TRAF6), leading to the decreased phosphorylation levels of NF-κB upstream regulators such as IκB and IκB kinase (IKK). MC13 also significantly down-regulated the phosphorylation levels of ERK and p38 MAPKs, which played key roles in microglia-mediated inflammatory response. Furthermore, MC13 inhibited Jak2-dependent Stat1/3 signaling pathway activation by blocking Jak2 phosphorylation, Stat1/3 phosphorylation, and nuclear translocation. Taken together, our results demonstrated that MC13 protected neurons from microglia-mediated neuroinflammatory injury by inhibiting TRAF6-TAK1-NF-κB, p38/ERK MAPKs, and Jak2-Stat1/3 pathways. Finally, MC13 might interact with LPS and interfere LPS-binding to cell membrane surface. These findings suggested that coumarin might act as a potential medicinal agent for treating neuroinflammation as well as inflammation-related neurodegenerative diseases.

Anti-inflammatory coumarin and benzocoumarin derivatives from Murraya alata.

Two new rare 8-methylbenzo[h]coumarins, muralatins A and B (1, 2), nine new C-8-substituted coumarins, muralatins C-K (3-11), and 22 known analogues (12-33) were isolated from the leaves of Murraya alata. The absolute configurations of compounds 5, 11, 23, 24, 27, 30, and 33 were assigned via comparison of their specific rotations, by Mosher's method, and by single-crystal X-ray diffraction and electronic circular dichroism (ECD) data of the in situ formed transition metal complexes. A putative biosynthesis pathway to 1 and 2 is proposed, and the chemical synthesis of 1 was accomplished through electrocyclization of 5,7-dimethoxy-8-[(Z)-3-methylbut-1,3-dienyl)]coumarin (12). Compounds 1, 2, 8, 12, and 31 showed inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages with IC50 values of 6.0-14.5 µM.

[Chemical constituents from roots of Illicium majus].

Ten compounds, including seven sesquiterpenes, two phenols and one phenylpropanoid, were isolated from the roots of Illicium majus by means of silica gel, ODS, Sephadex LH-20, and preparative HPLC. On analysis of MS and NMR spectroscopic data , their structures were established as cycloparviflorolide (1), cycloparvifloralone (2), tashironin (3), tashironin A (4), anislactone A(5), anislactone B (6), pseudomajucin (7), syringaldehyde (8), methyl-4-hydroxy-3, 5-dimethoxybenzoate (9), and (E)-3-methoxy-4,5-methylenedioxycinnamic alchol (10), respectively. Compounds 1-4 and 8-10 were first isolated from this plant. In the in vitro assays, at a concentration of 1.0 x 10(-5) mol x L(-1), compounds 5 and 6 were active against LPS induced NO production in microglia with a inhibition rate of 75.31% and 53.7%, respectively.