The ethanolic extract of Curcuma longa grown in Korea exhibits anti-neuroinflammatory effects by activating of nuclear transcription factor erythroid-2-related factor 2/heme oxygenase-1 signaling pathway.

BACKGROUND: Curcuma longa has been used as spices, food preservative, coloring material, and traditional medicine. This plant also has long been used for a variety of diseases including dyslipidemia, stomach disorders, arthritis, and hepatic diseases. The aim of the present investigation was to examine the anti-neuroinflammatory effects of the 50% ethanolic extract of C. longa in lipopolysaccharide (LPS)-induced BV2 microglial cells. METHODS: Griess reaction was employed to measure the production of nitric oxide (NO), and the levels of prostaglandin E2 (PGE2) and pro-inflammatory cytokines such as interleukin 1-beta (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) were determined by using profit ELISA kits. Western blotting was used to determine the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), mitogen activated protein kinases (MAPKs), heme oxygenase-1 (HO-1) and nuclear factor erythroid-2-related factor 2 (Nrf2). RESULTS: Pre-treatment with CLE inhibited the overproduction and overexpression of pro-inflammatory mediators including NO, PGE2, iNOS, COX-2, and pro-inflammatory cytokines such as IL-1ß, IL-6 and TNF-α in LPS-induced BV2 cells. In addition, CLE suppressed the activation of the NF-κB and three MAPK signaling pathways. Treatment with CLE induced HO-1 protein expression by activating Nrf2 pathway, and inhibiting the HO-1 expression reversed the anti-inflammatory effect of CLE. CONCLUSION: CLE showed anti-neuroinflammatory effects against LPS-induced microglial cells activation through the inhibition of production and expression of pro-inflammatory mediators by negative regulation of the NF-κB and MAPK signaling pathways. These anti-neuroinflammatory effects of CLE were mediated by HO-1/Nrf2 signaling pathway. Taken together, the present study suggests a potent effect of CLE to prevent neuroinflammatory diseases. It is necessary to perform additional efficacy evaluation through in vivo experiments.

Pivotal role of PD-1/PD-L1 immune checkpoints in immune escape and cancer progression: Their interplay with platelets and FOXP3+Tregs related molecules, clinical implications and combinational potential with phytochemicals.

Immune checkpoint proteins including programmed cell death protein 1 (PD-1), its ligand PD-L1 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) are involved in proliferation, angiogenesis, metastasis, chemoresistance via immune escape and immune tolerance by disturbing cytotoxic T cell activation. Though many clinical trials have been completed in several cancers by using immune checkpoint inhibitors alone or in combination with other agents to date, recently multi-target therapy is considered more attractive than monotherapy, since immune checkpoint proteins work with other components such as surrounding blood vessels, dendritic cells, fibroblasts, macrophages, platelets and extracellular matrix within tumor microenvironment. Thus, in the current review, we look back on research history of immune checkpoint proteins and discuss their associations with platelets or tumor cell induced platelet aggregation (TCIPA) and FOXP3+ regulatory T cells (Tregs) related molecules involved in immune evasion and tumor progression, clinical implications of completed trial results and signaling networks by phytochemicals for combination therapy with immune checkpoint inhibitors and suggest future research perspectives.

Whole-genome, transcriptome, and methylome analyses provide insights into the evolution of platycoside biosynthesis in Platycodon grandiflorus, a medicinal plant.

Triterpenoid saponins (TSs) are common plant defense phytochemicals with potential pharmaceutical properties. Platycodon grandiflorus (Campanulaceae) has been traditionally used to treat bronchitis and asthma in East Asia. The oleanane-type TSs, platycosides, are a major component of the P. grandiflorus root extract. Recent studies show that platycosides exhibit anti-inflammatory, antiobesity, anticancer, antiviral, and antiallergy properties. However, the evolutionary history of platycoside biosynthesis genes remains unknown. In this study, we sequenced the genome of P. grandiflorus and investigated the genes involved in platycoside biosynthesis. The draft genome of P. grandiflorus is 680.1 Mb long and contains 40,017 protein-coding genes. Genomic analysis revealed that the CYP716 family genes play a major role in platycoside oxidation. The CYP716 gene family of P. grandiflorus was much larger than that of other Asterid species. Orthologous gene annotation also revealed the expansion of ß-amyrin synthases (bASs) in P. grandiflorus, which was confirmed by tissue-specific gene expression. In these expanded gene families, we identified key genes showing preferential expression in roots and association with platycoside biosynthesis. In addition, whole-genome bisulfite sequencing showed that CYP716 and bAS genes are hypomethylated in P. grandiflorus, suggesting that epigenetic modification of these two gene families affects platycoside biosynthesis. Thus whole-genome, transcriptome, and methylome data of P. grandiflorus provide novel insights into the regulation of platycoside biosynthesis by CYP716 and bAS gene families.

Cartilage protective and anti-analgesic effects of ALM16 on monosodium iodoacetate induced osteoarthritis in rats.

BACKGROUND: Osteoarthritis (OA) is an age-related joint disease with characteristics that involve the progressive degradation of articular cartilage and resulting chronic pain. Previously, we reported that Astragalus membranaceus and Lithospermum erythrorhizon showed significant anti-inflammatory and anti-osteoarthritis activities. The objective of this study was to examine the protective effects of ALM16, a new herbal mixture (7:3) of ethanol extracts of A. membranaceus and L. erythrorhizon, against OA in in vitro and in vivo models. METHODS: The levels of matrix metalloproteinase (MMP)-1, -3 and - 13 and glycosaminoglycan (GAG) in interleukin (IL)-1ß or ALM16 treated SW1353 cells were determined using an enzyme-linked immunosorbent and quantitative kit, respectively. In vivo, the anti-analgesic and anti-inflammatory activities of ALM16 were assessed via the acetic acid-induced writhing response and in a carrageenan-induced paw edema model in ICR mice, respectively. In addition, the chondroprotective effects of ALM16 were analyzed using a single-intra-articular injection of monosodium iodoacetate (MIA) in the right knee joint of Wister/ST rat. All samples were orally administered daily for 2 weeks starting 1 week after the MIA injection. The paw withdrawal threshold (PWT) in MIA-injected rats was measured by the von Frey test using the up-down method. Histopathological changes of the cartilage in OA rats were analyzed by hematoxylin and eosin (H&E) staining. RESULTS: ALM16 remarkably reduced the GAG degradation and MMP levels in IL-1ß treated SW1353 cells. ALM16 markedly decreased the thickness of the paw edema and writhing response in a dose-dependent manner in mice. In the MIA-induced OA rat model, ALM16 significantly reduced the PWT compared to the control group. In particular, from histological observations, ALM16 showed clear improvement of OA lesions, such as the loss of necrotic chondrocytes and cartilage erosion of more than 200 mg/kg b.w., comparable to or better than a positive drug control (JOINS™, 200 mg/kg) in the cartilage of MIA-OA rats. CONCLUSIONS: Our results demonstrate that ALM16 has a strong chondroprotective effect against the OA model in vitro and in vivo, likely attributed to its anti-inflammatory activity and inhibition of MMP production.

Ginseng Berry Prevents Alcohol-Induced Liver Damage by Improving the Anti-Inflammatory System Damage in Mice and Quality Control of Active Compounds.

The ginseng berry contains a variety of biologically active compounds and has a higher ginsenoside content than its roots. This study focused on the hepatoprotective activity of ginseng berry extract prepared by enzyme treatment (EGB) compared to the non-enzyme-treated ginseng berry extract (GB) and quality control of EGB. The feeding effect of EGB on alcohol-induced liver damage (AILD) was investigated by measuring the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) compared with those of EtOH-fed mice. Furthermore, cytokine levels in the culture supernatants of EGB- or GB-treated RAW 264.7 cells were determined by enzyme-linked immunosorbent assay. The developed method was applied to the simultaneous quantification of four major ginsenosides in EGB using UPLC-QTOF/MS. Treatment with EGB at a dose of 0.5 or 1 mg/mouse significantly suppressed the AST and ALT levels in mice with AILD. Enzyme-treated ginseng berry was also found to suppress the production of inflammatory mediators like nitric oxide (NO), tumor-necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, showing higher activity than that of GB. The amount of ginsenoside Re, F5, F3, and Rd in the EGB obtained using UPLC-QTOF/MS was 45.9, 3.3, 4.0, and 6.2 mg/g, respectively. These results suggest that EGB has a potential effect on AILD, and its hepatoprotective effect provides beneficial insights into developing new candidates for the prevention and cure of AILD. Also, this study demonstrated the utility of UPLC-QTOF/MS-based major compounds for quality control (QC) of EGB.

Antihypertensive Effect of Ethanolic Extract from Acanthopanax sessiliflorus Fruits and Quality Control of Active Compounds.

Acanthopanax sessiliflorus (Rupr. & Maxim.) Seem., which belongs to the Araliaceae family, mainly inhabits Korea, China, and Japan. Traditionally, Acanthopanax species have been used as treatment for several diseases such as diabetes, tumors, and rheumatoid arthritis. Especially, its fruits have many biological functions including antitumor, immunostimulating, antithrombosis, and antiplatelet activities. Recently, the extract of A. sessiliflorus fruit has been reported to have antithrombotic and antiplatelet activities related to the alleviation of hypertension. Therefore, we investigated the antihypertensive effect of ethanolic extract from A. sessiliflorus fruits (DHP1501) through in vivo, ex vivo, and in vitro studies. In this study, DHP1501 demonstrated free radical scavenging capacity, enhanced endothelial nitric oxide (NO) production, and inhibited angiotensin-converting enzyme (ACE) activity in spontaneously hypertensive rats (SHRs), resulting in the improvement of vascular relaxation and decrease in blood pressure in the hypertensive animal model. These results suggest that A. sessiliflorus fruit extract may be a promising functional material for the prevention and treatment of hypertension. Furthermore, this study demonstrated the utility of MS-based active compounds for the quality control of DHP1501.

Isolation and Quantification of Ginsenoside Rh23, a New Anti-Melanogenic Compound from the Leaves of Panax ginseng.

A new ginsenoside, named ginsenoside Rh23 (1), and 20-O-ß-d-glucopyranosyl-3ß,6α,12ß,20ß,25-pentahydroxydammar-23-ene (2) were isolated from the leaves of hydroponic Panax ginseng. Compounds were isolated by various column chromatography and their structures were determined based on spectroscopic methods, including high resolution quadrupole/time of flight mass spectrometry (HR-QTOF/MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. To determine anti-melanogenic activity, the change in the melanin content in melan-a cells treated with identified compounds was tested. Additionally, we investigated the melanin inhibitory effects of ginsenoside Rh23 on pigmentation in a zebrafish in vivo model. Compound 1 inhibited potent melanogenesis in melan-a cells with 37.0% melanogenesis inhibition at 80 µM and also presented inhibition on the body pigmentation in zebrafish model. Although compound 2 showed slightly lower inhibitory activity than compound 1, it also showed significantly decreased melanogenesis in melan-a cell and in zebrafish model. These results indicated that compounds isolated from hydroponic P. ginseng may be used as new skin whitening compound through the in vitro and in vivo systems. Furthermore, this study demonstrated the utility of MS-based compound 1 for the quantitative analysis. Ginsenoside Rh23 (1) was found at a level of 0.31 mg/g in leaves of hydroponic P. ginseng.

Pseudoshikonin I enhances osteoblast differentiation by stimulating Runx2 and Osterix.

Pseudoshikonin I (PSI), a novel biomaterial isolated from Lithospermi radix, has been recognized as an herbal medicine for the treatment of infectious and inflammatory diseases. Bone remodeling maintains a balance through bone resorption (osteoclastogenesis) and bone formation (osteoblastogenesis). Bone formation is generally attributed to osteoblasts. However, the effects of PSI on the bone are not well known. In this study, we found that the ethanol extracts of PSI induced osteoblast differentiation by increasing the expression of bone morphogenic protein 4 (BMP 4). PSI positively regulates the transcriptional expression and osteogenic activity of osteoblast-specific transcription factors such as Runx2 and Osterix. To identify the signaling pathways that mediate PSI-induced osteoblastogenesis, we examined the effects of serine-threonine kinase inhibitors that are known regulators of Osterix and Runx2. PSI-induced upregulation of Osterix and Runx2 was suppressed by treatment with AKT and PKA inhibitors. These results suggest that PSI enhances osteoblast differentiation by stimulating Osterix and Runx2 via the AKT and PKA signaling pathways. Thus, the activation of Runx2 and Osterix is modulated by PSI, thereby demonstrating its potential as a treatment target for bone disease.

Characterization of the changes in eicosanoid profiles of activated macrophages treated with 20(S)-ginsenoside Rg3.

In this study, we used ultra-performance liquid chromatography coupled with tandem mass spectrometry to assess the levels of eicosanoids from RAW264.7 macrophages treated with lipopolysaccharides (LPS) and 20(S)-ginsenoside Rg3 (Rg3). The production of nitric oxide (NO) and the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were increased in inflammatory macrophages treated with LPS. Rg3 treatment, however, decreased the levels of NO, TNF-α, and IL-6 in activated macrophages. Eicosanoids, known as major metabolites correlated with inflammation, have pro- or anti-inflammatory activities. For a detailed characterization of the eicosanoids altered by treatment with LPS and Rg3, the eicosanoids were profiled by multiple reaction monitoring. A total of 69 macrophage eicosanoids were analyzed and the profiling dataset was statistically analyzed. Principal component and hierarchical cluster analyses differentiated control cells from cells treated with LPS, Rg3, or LPS+Rg3 for 12 or 24h. Furthermore, 18 differentially regulated eicosanoids were found between macrophages treated with LPS for 24h and those treated with LPS+Rg3 for 24h (fold change>2, p value<0.05). These results indicate that Rg3 alters eicosanoid metabolism in activated macrophages treated with LPS. Furthermore, we also identified several eicosanoids correlated with the anti-inflammatory activity of Rg3.

UPLC-QqQ/MS-Based Lipidomics Approach To Characterize Lipid Alterations in Inflammatory Macrophages.

In this study, UPLC-QqQ/MS-based lipidomics was applied to profile various lipids from RAW264.7 macrophages treated with different concentrations of lipopolysaccharide (LPS). The degree of inflammation increased with the LPS concentration. To elucidate the altered lipid metabolism of inflammatory macrophages, we targeted to analyze 25 lipid classes from LPS-treated RAW264.7 cells. As a result, 523 lipid species were successfully profiled by using the optimal UPLC and MRM. Statistical data analyses such as PCA, PLS-DA, and HCA differentiated five RAW264.7 cells treated with different concentrations of LPS. VIP plot, heat map, and bar plot also provided lists of up- or down-regulated lipids according to the LPS concentration. From the results, 11 classes of lipids, TG, DG, ChE, PE, PS, PI, PA, LyPC, LyPE, Cer, and dCer, were increased, and three classes, cholesterol, PC, and LyPA, were decreased in an LPS concentration-dependent manner. Furthermore, the treatment of an anti-inflammatory compound recovered the levels of PC, PE, PI, PA, LyPE, LyPA, and Cer from the activated macrophages. Finally, these results demonstrate the correlation between inflammation and lipid metabolism in macrophages. The differentially regulated lipids also have the potential to be used as biomarkers for inflammation.

Potential of Pseudoshikonin I Isolated from Lithospermi Radix as Inhibitors of MMPs in IL-1ß-Induced SW1353 Cells.

Pseudoshikonin I, the new bioactive constituent of Lithospermi radix, was isolated from this methanol extract by employing reverse-phase medium-pressure liquid chromatography (MPLC) using acetonitrile/water solvent system as eluents. The chemical structure was determined based on spectroscopic techniques, including 1D NMR (¹H, (13)C, DEPT), 2D NMR (gCOSY, gHMBC, gHMQC), and QTOF/MS data. In this study, we demonstrated the effect of pseudoshikonin I on matrix-metalloproteinase (MMPs) activation and expression in interleukin (IL)-1ß-induced SW1353 chondrosarcoma cells. MMPs are considered important for the maintenance of the extracellular matrix. Following treatment with PS, active MMP-1, -2, -3, -9, -13 and TIMP-2 were quantified in the SW1353 cell culture supernatants using a commercially available ELISA kit. The mRNA expression of MMPs in SW1353 cells was measured by RT-PCR. Pseudoshikonin I treatment effectively protected the activation on all tested MMPs in a dose-dependent manner. TIMP-2 mRNA expression was significantly upregulated by pseudoshikonin I treatment. Overall, we elucidated the inhibitory effect of pseudoshikonin on MMPs, and we suggest its use as a potential novel anti-osteoarthritis agent.

Ethanol extract of Lithospermum erythrorhizon Sieb. et Zucc. promotes osteoblastogenesis through the regulation of Runx2 and Osterix.

Bone remodeling and homeostasis are largely the result of the coordinated action of osteoblasts and osteoclasts. Osteoblasts are responsible for bone formation. The differentiation of osteoblasts is regulated by the transcription factors, Runx2 and Osterix. Natural products of plant origin are still a major part of traditional medicinal systems in Korea. The root of Lithospermum erythrorhizon Sieb. et Zucc. (LR), the purple gromwell, is an herbal medicine used for inflammatory and infectious diseases. LR is an anti-inflammatory and exerts anticancer effects by inducing the apoptosis of cancer cells. However, the precise molecular signaling mechanisms of osteoblastogenesis as regards LR and osteoblast transcription are not yet known. In this study, we investigated the effects of ethanol (EtOH) extract of LR (LES) on the osteoblast differentiation of C2C12 myoblasts induced by bone morphogenetic protein 4 (BMP4) and the potential involvement of Runx2 and Osterix in these effects. We found that the LES exhibited an ability to induce osteoblast differentiation. LES increased the expression of the osteoblast marker, alkaline phosphatase (ALP), as well as its activity, as shown by ALP staining and ALP activity assay. LES also increased mineralization, as shown by Alizarin Red S staining. Treatment with LES increased the protein levels (as shown by immunoblotting), as well as the transcriptional activity of Runx2 and Osterix and enhanced osteogenic activity. These results suggest that LES modulates osteoblast differentiation at least in part through Runx2 and Osterix.

A synthetic chalcone derivative, 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139), suppresses the TNFα-induced invasive capability of MDA-MB-231 human breast cancer cells by inhibiting NF-κB-mediated GROα expression.

2-Hydroxy-3',5,5'-trimenthoxyochalcone (DK-139) is a synthetic chalcone-derived compound. This study evaluated the biological activity of DK-139 on the inhibition of tumor metastasis. Growth-regulated oncogene-alpha (GROα) plays an important role in the progression of tumor development by stimulating angiogenesis and metastasis. In this study, DK-139 inhibited tumor necrosis factor alpha (TNFα)-induced GROα gene promoter activity by inhibiting of IκB kinase (IKK) in MDA-MB231 cells. In addition, DK-139 prevented the TNFα-induced cell migration, F-actin formation, and invasive capability of MDA-MB-231 cells. These findings suggest that DK-139 is a potential drug candidate for the inhibition of tumor cell locomotion and invasion via the suppression of NF-κB-mediated GROα expression.

The Mechanism Underlying the Antibacterial Activity of Shikonin against Methicillin-Resistant Staphylococcus aureus.

Shikonin (SKN), a highly liposoluble naphthoquinone pigment isolated from the roots of Lithospermum erythrorhizon, is known to exert antibacterial, wound-healing, anti-inflammatory, antithrombotic, and antitumor effects. The aim of this study was to examine SKN antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The SKN was analyzed in combination with membrane-permeabilizing agents Tris and Triton X-100, ATPase inhibitors sodium azide and N,N'-dicyclohexylcarbodiimide, and S. aureus-derived peptidoglycan; the effects on MRSA viability were evaluated by the broth microdilution method, time-kill test, and transmission electron microscopy. Addition of membrane-permeabilizing agents or ATPase inhibitors together with a low dose of SKN potentiated SKN anti-MRSA activity, as evidenced by the reduction of MRSA cell density by 75% compared to that observed when SKN was used alone; in contrast, addition of peptidoglycan blocked the antibacterial activity of SKN. The results indicate that the anti-MRSA effect of SKN is associated with its affinity to peptidoglycan, the permeability of the cytoplasmic membrane, and the activity of ATP-binding cassette (ABC) transporters. This study revealed the potential of SKN as an effective natural antibiotic and of its possible use to substantially reduce the use of existing antibiotic may also be important for understanding the mechanism underlying the antibacterial activity of natural compounds.

A direct protein kinase B-targeted anti-inflammatory activity of cordycepin from artificially cultured fruit body of Cordyceps militaris.

BACKGROUND: Cordyceps militaris is one of well-known medicinal mushrooms with anti-inflammatory, anti-cancer, anti-diabetic, and anti-obesity activities. OBJECTIVE: The objective of the following study is to isolate chemical components from the ethanol extract (Cm-EE) from Cordyceps militaris and to evaluate their anti-inflammatory activities. MATERIALS AND METHODS: Column chromatographic separation was performed and anti-inflammatory roles of these compounds were also examined by using NO production and protein kinase B (AKT) activity assays. RESULTS: From Cm-EE, 13 constituents, including trehalose (1), cordycepin (2), 6-hydroxyethyladenosine (3), nicotinic amide (4), butyric acid (5), ß-dimorphecolic acid (6), α-dimorphecolic acid (7), palmitic acid (8), linoleic acid (9), cordycepeptide A (10), 4-(2-hydroxy-3-((9E,12E)-octadeca-9,12-dienoyloxy)propoxy)-2-(trimethylammonio)butanoate (11), 4-(2-hydroxy-3-(palmitoyloxy)propoxy)-2-(trimethylammonio)butanoate (12), and linoleic acid methyl ester (13) were isolated. Of these components, compound 2 displayed a significant inhibitory effect on NO production in lipopolysaccharide (LPS)-activated RAW264.7 cells. Furthermore, this compound strongly and directly suppressed the kinase activity of AKT, an essential signalling enzyme in LPS-induced NO production, by interacting with its ATP binding site. CONCLUSION: C. militaris could have anti-inflammatory activity mediated by cordycepin-induced suppression of AKT.

Glycosyl glycerides from hydroponic Panax ginseng inhibited NO production in lipopolysaccharide-stimulated RAW264.7 cells.

BACKGROUND: Although the aerial parts of hydroponic Panax ginseng are reported to contain higher contents of total ginsenosides than those of roots, the isolation and identification of active metabolites from the aerial parts of hydroponic P. ginseng have not been carried out so far. METHODS: The aerial parts of hydroponic P. ginseng were applied on repeated silica gel and octadecylsilane columns to yield four glycosyl glycerides (Compounds 1-4), which were identified based on nuclear magnetic resonance, infrared, fast atom bombardment mass spectrometry, and gas chromatography/mass spectrometry data. Compounds 1-4 were evaluated for inhibition activity on NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. RESULTS AND CONCLUSION: The glycosyl glycerides were identified to be (2S)-1-O-7(Z),10(Z),13(Z)-hexadecatrienoyl-3-O-ß-d-galactopyranosyl-sn-glycerol (1), (2S)-1-O-linolenoyl-3-O-ß-d-galactopyranosyl-sn-glycerol (2), (2S)-1-O-linolenoyl-2-O-linolenoyl-3-O-ß-d-galactopyranosyl-sn-glycerol (3), and 2(S)-1-O-linoleoyl-2-O-linoleoyl-3-O-ß-d-galactopyranosyl-sn-glycerol (4). Compounds 1 and 2 showed moderate inhibition activity on NO production in LPS-stimulated RAW264.7 cells [half maximal inhibitory concentration (IC50): 63.8 ± 6.4µM and 59.4 ± 6.8µM, respectively] without cytotoxicity at concentrations < 100µM, whereas Compounds 3 and 4 showed good inhibition effect (IC50: 7.7 ± 0.6µM and 8.0 ± 0.9µM, respectively) without cytotoxicity at concentrations < 20µM. All isolated compounds showed reduced messenger RNA (mRNA) expression of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α in LPS-induced macrophage cells with strong inhibition of mRNA activity observed for Compounds 3 and 4.

Anti-inflammatory activity of a new cyclic peptide, citrusin XI, isolated from the fruits of Citrus unshiu.

ETHNOPHARMACOLOGICAL RELEVANCE: Citrus unshiu (Rutaceae) is an easy-peeling citrus fruit, which has been used as a traditional Korean medicine for improving skin elasticity, relieving fatigue and cough, and preventing bronchitis, flu, and various cancers. However, its active components associated with anti-inflammation and underlying mechanisms remain unknown. In this study, we investigated the active constituents from the fruits of Citrus unshiu and evaluated the anti-inflammatory activity in order to support the traditional usage of Citrus unshiu. MATERIAL AND METHODS: Repeated column chromatography, together with a semi-preparative HPLC purification was used to separate the bioactive constituent from the EtOAc soluble fraction of the EtOH extract of Citrus unshiu fruits. Anti-inflammatory effects of the isolated compounds on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW264.7 macrophage cells. RESULTS: A new cyclic peptide, citrusin XI (1), was isolated and identified from the fruits of Citrus unshiu. The structure of compound 1 was elucidated by spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) ((1)H, (13)C, COSY, HMQC and HMBC experiments), and high resolution (HR)-mass spectrometry, and its absolute configurations were further confirmed by the Marfey׳s method. Compound 1 decreased NO production in LPS-stimulated RAW264.7 cells in a dose-dependent manner with an IC50 value of 70µM. Compound 1 suppressed NO production by decreasing iNOS expression but COX-2 expression was slightly associated with the reduction by compound 1 in LPS-induced RAW264.7 cells. Furthermore, compound 1 inhibited NF-κB activation by blocking IκBα degradation and NF-κB phosphorylation in LPS-stimulated RAW264.7 cells. CONCLUSIONS: These results indicate that a new cyclic peptide, citrusin XI, from Citrus unshiu fruits has anti-inflammatory properties that inhibit the release of pro-inflammatory mediators. Compound 1 decreases NO production by decreasing iNOS expression and NF-κB activation associated with IκBα degradation and NF-κB phosphorylation in LPS-induced RAW264.7 cells. This is the first study to clarify the underlying mechanism of the anti-inflammatory effect exerted by a pure isolated compound from Citrus unshiu in LPS-stimulated RAW264.7 macrophage cells. The phytochemical, citrusin XI of Citrus unshiu may serve as lead compound in the design of new agents for preventing and treating inflammatory diseases.

2-Hydroxy-3,4-naphthochalcone (2H-NC) inhibits TNFα-induced tumor invasion through the downregulation of NF-κB-mediated MMP-9 gene expression.

The control of tumor metastasis is important for the successful prevention and treatment of cancer. Emerging evidence indicates that various natural and synthetic chalcones exhibit antimetastatic activity through the inhibition of nuclear factor-κB (NF-κB), although the precise mechanism by which this occurs is currently unclear. In this study, 2-hydroxy-3,4-naphthochalcone (2H-NC) was found to reduce tumor necrosis factor alpha (TNFα)-induced MMP-9 mRNA expression and gelatinolytic enzyme activity. These actions were associated with inhibition of RelA/p65 NF-κB activity. In addition, 2H-NC inhibited TNFα-induced invasion of MDA-MB-231 breast cancer cells, as assessed using a three-dimensional spheroid invasion assay. Taken together, these data demonstrate that 2H-NC prevents TNFα-induced tumor cell invasion through downregulation of NF-κB-mediated MMP-9 gene expression, and thereby identify naphthochalcones as a potentially effective class of molecules to use as a platform for the development of antimetastatic agents.

Neolignans from the fruits of Magnolia obovata and their inhibition effect on NO production in LPS-induced RAW 264.7 cells.

Three new neolignans, named 9-methoxyobovatol (6), magnobovatol (7), and 2-hydroxyobovaaldehyde (9), along with six known ones, magnolol (1), honokiol (2), isomagnolol (3), obovatol (4), obovatal (5), and obovaaldehyde (8), were isolated from the fruits of Magnolia obovata using silica gel and ODS column chromatography. From the results of spectroscopic data including EIMS, IR, 1H- and 13C-NMR, DEPT, and 2D-NMR (gCOSY, gHSQC, gHMBC), the chemical structures were determined. All isolated compounds were evaluated for inhibition activity on nitric oxide production in LPS-induced RAW 264.7 cells, and compounds 1-4, 6, 7, and 9 showed significant activity with IC50 values of 15.8 ± 0.3, 3.3 ± 1.2, 14.1 ± 0.9, 6.2 ± 1.2, 14.8 ± 2.3, 14.2 ± 1.2, and 14.8 ± 3.2 µM, respectively, without any visible toxic effect.

Phenolic components from Rhus parviflora fruits and their inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages.

Nine phenolic compounds, phloracetophenone-4-O-ß-D-glucopyranoside (1), p-hydroxybenzoic acid-4-O-ß-D-glucopyranoside (2), leonuriside A (3), 3-methoxy-4-hydroxyphenol-1-O-ß-D-glucopyranoside (4), cis-p-coumaric acid-4-O-ß-D-glucopyranoside (5), trans-p-coumaric acid-4-O-ß-D-glucopyranoside (6), trans-p-coumaric acid-9-O-ß-D-glucopyranoside (7), (-)-shikimic acid (8) and (-)-methyl shikimate (9), were isolated for the first time from the fruits of Rhus parviflora. Compounds 1, 3-6 and 8 inhibited lipopolysaccharide-stimulated nitric oxide (NO) production and inducible NO synthase expression in RAW 264.7 macrophages with IC50 values of 9.24 ± 1.20, 21.37 ± 2.02, 23.07 ± 1.58, 9.86 ± 0.98, 19.05 ± 1.66 and 11.3 ± 1.54 µM, respectively. The results indicated possible use of compounds for the treatment of inflammatory diseases.