Bioactivity-based analysis and chemical characterization of anti-inflammatory compounds from Curcuma zedoaria rhizomes using LPS-stimulated RAW264.7 cells.

Inflammation is not only a self-defense response of the innate immune system, but also the pathogenesis mechanism of multiple diseases such as arthritis, neurodegeneration, and cancer. Curcuma zedoaria Roscoe (Zingiberaceae), an indigenous plant of India, has been used traditionally in Ayurveda and folk medicine. As part of our ongoing efforts to screen traditional medicinal plants exhibiting pharmacological potential and to characterize the compounds involved, we examined the anti-inflammatory effects of the MeOH extract of C. zedoaria rhizomes using lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage cells and found that MeOH extract inhibited the synthesis of nitric oxide (NO) in a dose-dependent manner (IC50: 23.44 ±â€¯0.77 µg/mL). In our efforts to characterize the compounds responsible for these anti-inflammatory effects, bioactivity-guided fractionation of the MeOH extract and chemical investigation of its active hexane-soluble fraction led to the successful isolation of five sesquiterpenes (1-5), the structures of which were elucidated by NMR spectroscopic analysis and LC/MS analysis. Among them, curcuzedoalide (5) exhibited potent inhibitory effects on NO synthesis (IC50: 12.21 ±â€¯1.67 µM) and also suppressed pre-inflammatory protein expression of iNOS and COX-2. Curcuzedoalide (5) was thus determined to be a contributor to the anti-inflammatory effect of C. zedoaria rhizomes and could be a potential candidate for therapeutic applications.

(-)-9'-O-(α-l-Rhamnopyranosyl)lyoniresinol from Lespedeza cuneata suppresses ovarian cancer cell proliferation through induction of apoptosis.

Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae), known as Chinese bushclover or sericea lespedeza, has been used in traditional medicine to treat diabetes, hematuria, and insomnia, and it has been reported that bioactive compounds from L. cuneata possess various pharmacological properties. However, there has been no study to determine the active compounds from L. cuneata with potential activity against ovarian cancer. This study aimed to isolate cytotoxic compounds from L. cuneata and identify the molecular mechanisms underlying the apoptosis pathway in ovarian cancer cells. Based on cytotoxic activity identified in the screening test, chemical investigation of the active fraction of L. cuneata led to the isolation of nine compounds including four lignanosides (1-4), three flavonoid glycosides (5-7), and two phenolics (8-9). Cytotoxicity and the molecular mechanism were examined by methyl thiazolyl tetrazolium (MTT) assay and Western blot analysis. Of the isolated compounds, (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol (3) demonstrated the strongest effect in suppressing A2780 human ovarian carcinoma cell proliferation in a dose-dependent manner, with an IC50 value of 35.40 ±â€¯2.78 µM. Control A2780 cells had normal morphology, whereas cell blebbing, shrinkage, and condensation were observed after treatment with compound 3. Western blotting analysis showed that compound 3 inhibited A2780 human ovarian cancer cell viability by activating caspase-8, caspase-3, and PARP, which contributed to apoptotic cell death. These results suggest that (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol (3) has potent anticancer activities against A2780 human ovarian carcinoma cells through the extrinsic apoptotic pathway. Therefore, (-)-9'-O-(α-l-rhamnopyranosyl)lyoniresinol is an excellent candidate for the development of novel chemotherapeutics.

Bioactive compounds from sclerotia extract of Poria cocos that control adipocyte and osteoblast differentiation.

Poria cocos Wolf confers edible sclerotia also known as 'Indian bread' in North America, that have been used for the treatment of various diseases in Asian countries. As part of our ongoing aim to identify biologically new metabolites from Korean edible mushrooms, we investigated the ethanol (EtOH) extract of the sclerotia of P. cocos by applying a comparative LC/MS- and bioassay-based analysis approach, since the EtOH extract reciprocally regulated adipocyte and osteoblast differentiation in mouse mesenchymal stem cells (MSCs). Bioassay-based analysis of the EtOH extract led to the successful isolation of two sterols, ergosterol peroxide (1) and 9,11-dehydroergosterol peroxide (2); three diterpenes, dehydroabietic acid (3), 7-oxocallitrisic acid, (4) and pimaric acid (5); and two triterpenes, dehydroeburicoic acid monoacetate (6) and eburicoic acid acetate (7) from the active hexane-soluble fraction. The isolated compounds (1-7) were examined for their effects on the regulation of MSC differentiation. The two sterols (1 and 2) were able to suppress MSC differentiation toward adipocytes. In contrast, the three diterpenes (3-5) showed activity to promote osteogenic differentiation of MSC. These findings demonstrate that the EtOH extract of P. cocos sclerotia is worth consideration as a new potential source of bioactive compounds effective in the treatment of osteoporosis in the elderly, since the extract contains sterols that inhibit adipogenic differentiation as well as diterpenes that promote osteogenic differentiation from MSCs.

A new cerebroside from the twigs of Lindera glauca (Sieb. et Zucc.) Blume.

Lindera glauca (Sieb. et Zucc.) Blume (Lauraceae) has been used to treat rheumatic arthritis, stroke, and cardiac pain. Phytochemical investigation of twigs of L. glauca (Sieb. et Zucc.) Blume resulted in the isolation and identification of a new cerebroside, glaucerebroside (1). The structure of 1 was elucidated by a combination of extensive spectroscopic analyses, including extensive 2D NMR, HR-MS, chemical reactions, and LC/MS analysis. Compound 1 is a relatively rare cerebroside with l-threo-configuration of the sphingosine part. This is the second example of identification of a cerebroside from the family Lauraceae. Compound 1 significantly inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV-2 cells, with an IC50 value of 23.84µM without inducing cell toxicity. This study suggests that glaucerebroside (1) can be an excellent candidate for development of novel anti-neuroinflammatory agents.

Bioactivity-guided isolation of anti-inflammatory triterpenoids from the sclerotia of Poria cocos using LPS-stimulated Raw264.7 cells.

Poria cocos Wolf (Polyporaceae) has been used as a medicinal fungus to treat various diseases since ancient times. This study aimed to investigate the anti-inflammatory chemical constituents of the sclerotia of P. cocos. Based on bioassay-guided fractionation using lipopolysaccharide (LPS)-stimulated Raw264.7 cells, chemical investigation of the EtOH extract of the sclerotia of P. cocos resulted in the isolation and identification of eight compounds including six triterpenoids, namely poricoic acid A (1), 3-O-acetyl-16α-hydroxydehydrotrametenolic acid (2), polyporenic acid C (3), 3ß-hydroxylanosta-7,9(11),24-trien-21-oic acid (4), trametenolic acid (5), and dehydroeburicoic acid (6), as well as (-)-pinoresinol (7) and protocatechualdehyde (8). The structures of the isolated compounds were determined by spectroscopic analysis, including 1H and 13C NMR spectra, and LC/MS analysis. The anti-inflammatory activities of the isolates were evaluated by estimating their effect on the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated Raw264.7 as well as on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Compounds 1-5 inhibited NO production and iNOS expression in LPS-stimulated Raw264.7 cells. Among them, compound 1 exerted the highest anti-inhibitory activity and reduced PGE2 levels via downregulation of COX-2 protein expression. The findings of this study provide experimental evidence that the sclerotia of P. cocos are a potential source of natural anti-inflammatory agents for use in pharmaceuticals and functional foods. Furthermore, the most active compound 1, seco-lanostane triterpenoid, could be a promising lead compound for the development of novel anti-inflammatory agents.

Lignans from the Twigs of Euonymus alatus (Thunb.) Siebold and Their Biological Evaluation.

A new sesquilignan, euonymolin A (1), and six known lignans, (-)-de-O-methylmagnolin (2), (+)-de-O-methylepimagnolin A (3), (+)-syringaresinol (4), (+)-pinoresinol (5), (+)-medioresinol (6), and (+)-lariciresinol 4'-O-ß-d-glucopyranoside (7), were isolated from the twigs of Euonymus alatus (Thunb.) Siebold (Celastraceae). The structures of the isolated compounds were elucidated based on spectroscopic analyses, including extensive 1D- and 2D-NMR techniques, HR-MS analysis and circular dichroism (CD) data, and the literature data. All of the isolated compounds were evaluated for antiproliferative activity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines and inhibition of nitric oxide (NO) production in a lipopolysaccharide (LPS)-activated BV2 cell line. All compounds showed cytotoxicity against the SK-MEL-2 cell line with IC50 values of 23.24 - 48.14 µm and inhibited NO production in LPS-activated BV-2 cells with IC50 values of 6.75 - 23.53 µm.

Anti-inflammatory and antitumor phenylpropanoid sucrosides from the seeds of Raphanus sativus.

A bioassay-guided fractionation and chemical investigation of the MeOH extract of Raphanus sativus seeds resulted in the isolation and identification of eight phenylpropanoid sucrosides (1-8) including two new compounds, named raphasativuside A and B (1-2) from the most active CHCl3-soluble fraction. The structures of these new compounds were elucidated through spectral analysis, including extensive 2D-NMR data, and chemical reaction experiments. We evaluated the anti-inflammatory effects of 1-8 in lipopolysaccharide (LPS)-stimulated murine microglia BV2 cells. Compounds 2 and 5 exhibited significant inhibitory effect on nitric oxide production in LPS-activated BV-2 cells with IC50 values of 21.63 and 26.96 µM, respectively. All isolates were also evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15). Compounds 1-7 showed consistent cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with IC50 values of 6.71-27.92 µM. Additionally, the free-radical scavenging activity of 1-8 was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay where compounds 1, 3, and 4 scavenged DPPH radical strongly with IC50 values of 23.05, 27.10, and 29.63 µg/mL, respectively.

Exogenous S1P Exposure Potentiates Ischemic Stroke Damage That Is Reduced Possibly by Inhibiting S1P Receptor Signaling.

Initial and recurrent stroke produces central nervous system (CNS) damage, involving neuroinflammation. Receptor-mediated S1P signaling can influence neuroinflammation and has been implicated in cerebral ischemia through effects on the immune system. However, S1P-mediated events also occur within the brain itself where its roles during stroke have been less well studied. Here we investigated the involvement of S1P signaling in initial and recurrent stroke by using a transient middle cerebral artery occlusion/reperfusion (M/R) model combined with analyses of S1P signaling. Gene expression for S1P receptors and involved enzymes was altered during M/R, supporting changes in S1P signaling. Direct S1P microinjection into the normal CNS induced neuroglial activation, implicating S1P-initiated neuroinflammatory responses that resembled CNS changes seen during initial M/R challenge. Moreover, S1P microinjection combined with M/R potentiated brain damage, approximating a model for recurrent stroke dependent on S1P and suggesting that reduction in S1P signaling could ameliorate stroke damage. Delivery of FTY720 that removes S1P signaling with chronic exposure reduced damage in both initial and S1P-potentiated M/R-challenged brain, while reducing stroke markers like TNF-α. These results implicate direct S1P CNS signaling in the etiology of initial and recurrent stroke that can be therapeutically accessed by S1P modulators acting within the brain.

Antineuroinflammatory and Antiproliferative Activities of Constituents from Tilia amurensis.

As part of our ongoing search for bioactive constituents of natural Korean medicinal resources, we found in a preliminary study that the methanol (MeOH) extract from the trunks of Tilia amurensis RUPR. showed an inhibitory effect on nitric oxide (NO) production in an activated murine microglial cell line. A bioassay-guided fractionation and chemical investigation of the MeOH extract resulted in the isolation and identification of a new isoflavonoid glycoside, orobol 4'-O-ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranoside (1) and 16 known compounds (2-17). The structure of the new compound was determined by spectroscopic methods, i.e., one-dimensional (1D) and two-dimensional (2D)-NMR techniques and high resolution (HR)-MS, and chemical methods. The antineuroinflammatory activities of the isolated compounds were determined by measuring NO levels in the medium using murine microglial BV-2 cells. Among them, 12 compounds, including compound 1 (most active with an IC50 value of 23.42 µM), inhibited NO production in lipopolysaccharide-stimulated BV-2 cells. Moreover, compounds 1-4 showed moderate antiproliferative activities against the SK-MEL-2 cell line, with IC50 values ranging from 12.31 to 19.67 µM.

Bioactive lignan constituents from the twigs of Lindera glauca.

A bioassay-guided fractionation and chemical investigation of the MeOH extract from the twigs of Lindera glauca (SIEB. et ZUCC.) BLUME resulted in the isolation and identification of six lignans (1-6) including three new lignan derivatives, named linderuca A (1), B (2), and C (3). The structures of the new compounds (1-3) were determined on the basis of spectroscopic analyses, including two dimensional NMR and circular dichroism (CD) spectroscopy studies. The cytotoxic activities of the isolates (1-6) were evaluated by determining their inhibitory effects on human tumor cell lines. Compounds 1-5 showed antiproliferative activities against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with IC50 values of 7.79-29.42 µM. Based on the understanding that inflammation is a crucial cause of tumor progression, we also investigated the anti-inflammatory activities of the isolates (1-6) in the lipopolysaccharide-stimulated murine microglia BV-2 cell line by measuring nitric oxide (NO) levels. The new lignans (1-3) significantly inhibited NO production with IC50 values of 12.10, 9.48, and 9.87 µM, respectively, without cytotoxicity.

Complete genome sequence of strain Lactiplantibacillus plantarum beLP1 a potential probiotic strain with antimicrobial properties isolated from kimchi.

The complete genome of the potential probiotic Lactiplantibacillus plantarum strain beLP1, isolated from kimchi in South Korea, was sequenced using Illumina and PacBio technologies. The genome comprises one circular chromosome and one plasmid without antimicrobial resistance genes.

Quinic acid derivatives from Pimpinella brachycarpa exert anti-neuroinflammatory activity in lipopolysaccharide-induced microglia.

Five new quinic acid derivatives (1-5), together with 10 known quinic acid derivatives (6-15), were isolated from the MeOH extract of Pimpinella brachycarpa (Umbelliferae). Their structures were established on the basis of spectroscopic analyses including extensive 2D NMR studies (COSY, HMQC and HMBC). Isolated compounds 1-15 were evaluated for their inhibitory activities on nitric oxide (NO) production in an activated murine microglial cell line. Compounds 2, 3, 8 and 11 significantly inhibited NO production without high cell toxicity in lipopolysaccharide (LPS)-activated BV-2 cells, a microglia cell line (IC50=4.66, 12.52, 9.04 and 12.11 µM, respectively).

Steroidal constituents from the leaves of Hosta longipes and their inhibitory effects on nitric oxide production.

Hosta longipes (FR. et SAV.) MATSUMURA (Liliaceae) is an edible vegetable in Korea. This study was conducted with the aim of evaluating the potential of H. longipes as a functional food for the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In this respect, the study resulted in the identification of three new steroidal compounds, longipenane (1), longipenane 26-O-ß-d-glucopyranoside (2) and neogitogenin 3-O-α-l-rhamnopyranosyl-(1→2)-O-[ß-d-glucopyranosyl-(1→4)]-ß-d-galactopyranoside (3), along with two known steroidal saponins (4 and 5). The identification and structural elucidation of these compounds were based on 1D and 2D NMR measurements, high-resolution FAB mass spectroscopy (HR-FAB-MS), and chemical methods. A proinflammatory mediator, nitric oxide (NO), in murine microglial BV-2 cells was used to assess the anti-neuroinflammatory effect of the isolated compounds from H. longipes. Among them, compounds 4 and 5 showed strong inhibitory effects on NO production without high cell toxicity in lipopolysaccharide-activated BV-2 cells (IC50=17.66 and 13.16µM, respectively).

Anti-neuroinflammatory diarylheptanoids from the rhizomes of Dioscorea nipponica.

In a continuing search for bioactive constituents from Dioscoreaceae medicinal plants, two new cyclic diarylheptanoids, diosniponol A (1) and B (2), together with 10 known compounds (3-12) were isolated from the rhizomes of Dioscorea nipponica. The structures of these new compounds were determined by spectroscopic analyses, including extensive two-dimensional nuclear magnetic resonance, high-resolution mass spectrometry, and optical rotation. All isolated compounds 1-12 were evaluated for their effects on nitric oxide (NO) production in murine microglia cell line BV-2. Compounds 8 and 11 showed potent inhibitory activities on NO production (IC50 13.36 and 14.36 µM, respectively) without cell toxicity in lipopolysaccharide-activated BV-2 cells.

Lanostane triterpenoids from the mushroom Naematoloma fasciculare.

In our continuing search for structurally interesting and bioactive metabolites from Korean wild mushrooms, bioassay-guided fractionation and a chemical investigation of the MeOH extracts of the fruiting bodies of the mushroom Naematoloma fasciculare resulted in the isolation of four new lanostane triterpenoids (1-4), together with 11 known compounds (5-15). The structures of 1-5 were determined by a combination of 1D and 2D NMR and HRMS. The absolute configuration of the 3-hydroxy-3-methylglutaryl group as a side chain in 1 and 2 was determined by the alkaline methanolysis method. The full NMR data assignment of the known compound fasciculol G (5) is reported for the first time. Compounds 1-15 were tested for their antiproliferative activities against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15) and evaluated for their inhibitory effects on nitric oxide production in a lipopolysaccharide-activated murine microglial cell line.

Quinone derivatives from the rhizomes of Acorus gramineus and their biological activities.

A further phytochemical investigation of the rhizomes of Acorus gramineus afforded three new quinone derivatives (1-3), together with two known compounds (4 and 5). The identification and structural elucidation of these new compounds were based on 1D and 2D NMR (COSY, HMQC, HMBC and NOESY) and MS data. The absolute configurations were established on the basis of their circular dichroism (CD) data. To investigate the anti-neuroinflammatory effects of the isolated compounds (1-5), the nitric oxide (NO) production was evaluated in the lipopolysaccharide-activated microglia cell line, BV-2. Compounds (1-5) were also tested for their cytotoxicity against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15) in vitro by using the SRB assay.

Bioactive sesquiterpenes from the essential oil of Thuja orientalis.

A phytochemical investigation on the essential oil of Thuja orientalis resulted in the isolation and identification of three new sesquiterpenes, 3α-methoxy-4α-epoxythujopsane (1), Δ³,¹5-4ß-epoxythujopsene (2), and Δ³,4-thujopsen-2,15-diol (3), together with eight known sesquiterpenoids (4-11). The structures of these new compounds were elucidated based on spectroscopic data analyses including extensive 2D-NMR data and HR-ESIMS. The full assignments of ¹H and ¹³C NMR chemical shifts for thujopsadiene (4) were obtained by 2D-NMR for the first time. All compounds (1-11) showed antiproliferative activities against the SK-OV-3 and SK-MEL-2 cell lines with IC50 values of 5.85-28.64 µM. In addition, compounds 1, 3, 4, 7, 8, and 9 significantly inhibited nitric oxide production in lipopolysaccharide-activated BV-2 cells with IC50 values of 3.93-17.85 µM without cell toxicity.

Acacetin attenuates neuroinflammation via regulation the response to LPS stimuli in vitro and in vivo.

Under normal conditions in the brain, microglia play roles in homeostasis regulation and defense against injury. However, over-activated microglia secrete proinflammatory and cytotoxic factors that can induce progressive brain disorders, including Alzheimer's disease, Parkinson's disease and ischemia. Therefore, regulation of microglial activation contributes to the suppression of neuronal diseases via neuroinflammatory regulation. In this study, we investigated the effects of acacetin (5,7-dihydroxy-4'-methoxyflavone), which is derived from Robinia pseudoacacia, on neuroinflammation in lipopolysaccharide (LPS)-stimulated BV-2 cells and in animal models of neuroinflammation and ischemia. Acacetin significantly inhibited the release of nitric oxide (NO) and prostaglandin E(2) and the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV-2 cells. The compound also reduced proinflammatory cytokines, tumor necrosis factor-α and interleukin-1ß, and inhibited the activation of nuclear factor-κB and p38 mitogen-activated protein kinase. In an LPS-induced neuroinflammation mouse model, acacetin significantly suppressed microglial activation. Moreover, acacetin reduced neuronal cell death in an animal model of ischemia. These results suggest that acacetin may act as a potential therapeutic agent for brain diseases involving neuroinflammation.

Flavonoid glycosides from the leaves of Allium victorialis var. platyphyllum and their anti-neuroinflammatory effects.

Eight new flavonoid glycosides, named allivictoside A-H (1-8), together with twelve known flavonoids (9-20) were isolated from the leaves of Allium victorialis var. platyphyllum. The structures of 1-8 were determined by chemical and spectroscopic methods, including 1D, 2D NMR analyses and HR-MS. To evaluate the anti-neuroinflammatory activities of all isolates, we measured the secreted nitric oxide levels in murine microglia BV-2 cells stimulated by lipopolysaccharide. In this study, compounds 2, 6, 10, and 18 significantly inhibited nitric oxide production (IC(50) values of 20.67, 20.42, 21.48 and 19.80 µM, respectively) without cell toxicity. Therefore, we suggest that allivictoside B (2) and F (6), 3-O-ß-D-glucosyl-7-O-ß-D-(2-O-feruloyl)glucosylkaempferol (10) and quercetin 3-O-ß-d-glucopyranoside (18) may be considered as candidates for the treatment of diseases associated with neuroinflammation.

A substituted 3,4-dihydropyrimidinone derivative (compound D22) prevents inflammation mediated neurotoxicity; role in microglial activation in BV-2 cells.

A novel synthetic 3,4-dihydropyrimidinone derivative, compound D22 (ethyl 6-methyl-4-(3-phenoxyphenyl)-2-thioxo-3,4-dihydropyrimidine-5-carboxylate), was found to exert anti-inflammatory properties in lipopolysaccharide-stimulated microglial BV-2 cells. Compound D22 reduced the pro-inflammatory factors such as nitric oxide, prostaglandin E(2), tumor necrosis factor-α and interleukin-1ß. Moreover, it suppressed the expressions of inducible NO synthase and cyclooxygenase-2. Compound D22 inhibited the activation of mitogen-activated protein kinases. When compound D22-conditioned media from BV-2 cells were applied to N2a cells, neuronal cell death was inhibited via suppression of caspase-3 activation and regulation of Bcl-2 and Bax proteins expression. These results suggest that compound D22 may be useful for treating neurodegenerative diseases related with neuroinflammation.