Sulfur-containing spiroketals from Breynia disticha and evaluations of their anti-inflammatory effect.

Breynia spp. are a key source of sulfur-containing spiroketal glycosides with potential anti-inflammatory activity. In this study, three new sulfur-containing spiroketals - breynin J (1), epibreynin J (2), and probreynogenin (3) - along with four known compounds - probreynin I (4), phyllaemblic acid (5), breynin B (6), and epibreynin B (7) - were isolated from the roots of Breynia disticha. The structures of compounds 1-7 were elucidated by extensive 1D and 2D NMR spectroscopic analyses, including 1D total correlation spectroscopy (TOCSY), HSQC, HMBC, double quantum-filtered (DQF)-COSY, heteronuclear two-bond correlation (H2BC), and HSQC-TOCSY experiments, as well as high-resolution electrospray ionization HRESIMS analysis, and quantum chemical electronic CD calculations. Furthermore, the absolute configurations of sugar residues were determined by derivatization of the hydrolysates with ʟ-cysteine methyl ester and o-tolyl isothiocyanate followed by HPLC analysis. The anti-inflammatory effects of the isolated compounds were evaluated based on the mRNA levels of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells. Compounds 1, 2, 6, and 7 inhibited the increase in interleukin (IL)-1ß and IL-6 mRNA levels stimulated by LPS. Moreover, the most potent compound 7 was found to significantly inhibit the production of IL-1ß and IL-6 proteins, as revealed by the analysis of culture supernatants.

Antiandrogenic Effects of a Polyphenol in Carex kobomugi through Inhibition of Androgen Synthetic Pathway and Downregulation of Androgen Receptor in Prostate Cancer Cell Lines.

Prostate cancer (PC) represents the most common cancer disease in men. Since high levels of androgens increase the risk of PC, androgen deprivation therapy is the primary treatment; however this leads to castration-resistant PC (CRPC) with a poor prognosis. The progression to CRPC involves ectopic androgen production in the adrenal glands and abnormal activation of androgen signaling due to mutations and/or amplification of the androgen receptor (AR) as well as activation of androgen-independent proliferative pathways. Recent studies have shown that adrenal-derived 11-oxygenated androgens (11-ketotestosterone and 11-ketodihydrotestosterone) with potencies equivalent to those of traditional androgens (testosterone and dihydrotestosterone) are biomarkers of CRPC. Additionally, dehydrogenase/reductase SDR family member 11 (DHRS11) has been reported to be a 17ß-hydroxysteroid dehydrogenase that catalyzes the production of the 11-oxygenated and traditional androgens. This study was conducted to evaluate the pathophysiological roles of DHRS11 in PC using three LNCaP, C4-2 and 22Rv1 cell lines. DHRS11 silencing and inhibition resulted in suppression of the androgen-induced expression of AR downstream genes and decreases in the expression of nuclear AR and the proliferation marker Ki67, suggesting that DHRS11 is involved in androgen-dependent PC cell proliferation. We found that 5,7-dihydroxy-8-methyl-2-[2-(4-hydroxyphenyl)ethenyl]-4H-1-benzopyran-4-one (Kobochromone A, KC-A), an ingredient in the flowers of Carex kobomugi, is a novel potent DHRS11 inhibitor (IC50 = 0.35 µM). Additionally, KC-A itself decreased the AR expression in PC cells. Therefore, KC-A suppresses the androgen signaling in PC cells through both DHRS11 inhibition and AR downregulation. Furthermore, KC-A enhanced the anticancer activity of abiraterone, a CRPC drug, suggesting that it may be a potential candidate for the development of drugs for the prevention and treatment of CRPC.

Japanese Huperzia serrata extract and the constituent, huperzine A, ameliorate the scopolamine-induced cognitive impairment in mice.

Huperzia serrata has been used as a Chinese folk medicine for many years. It contains huperzine A, which has a protective effect against memory deficits in animal models; however, it is unclear if H. serrata extract exerts any effects in Alzheimer's disease (AD) models. We used H. serrata collected in Japan and determined its huperzine A content using HPLC. We determined its inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. H. serrata extract (30 mg/kg/day) and donepezil (10 mg/kg/day) were orally administrated for 7 days. After repeated administration, we performed the Y-maze and passive avoidance tests. H. serrata extract contained 0.5% huperzine A; H. serrata extract inhibited AChE, but not BuChE. H. serrata extract ameliorated cognitive function in mice. These results indicate that Japanese H. serrata extract ameliorates cognitive function deficits by inhibiting AChE. Therefore, H. serrata extract may be valuable for the prevention or treatment of dementia in AD.

Methoxylated Flavones from Casimiroa edulis La Llave Suppress MMP9 Expression via Inhibition of the JAK/STAT3 Pathway and TNFα-Dependent Pathways.

Matrix metalloproteinase 9 (MMP9), an MMP isozyme, plays a crucial role in tumor progression by degrading basement membranes. It has therefore been proposed that the pharmacological inhibition of MMP9 expression or activity could inhibit tumor metastasis. We previously isolated two novel methoxylated flavones, casedulones A and B, from the leaves and/or roots of Casimiroa edulis La Llave and determined that these casedulones have antitumor activity that acts via the reduction of MMP9. Here, we examined how these casedulones suppress lipopolysaccharide (LPS)-induced MMP9 expression in human monocytic THP-1 cells. The casedulones suppressed the LPS-induced signal transducer and activator of transcription 3 (STAT3) pathway, which participates in MMP9 induction. In addition, AG490 and S3I-201, inhibitors of Janus kinase (JAK) and STAT3, suppressed LPS-mediated MMP9 induction, suggesting that the casedulones suppressed MMP9 induction through the inhibition of JAK/STAT3 pathways. Based on the findings that cycloheximide, an inhibitor of de novo protein synthesis, completely inhibited LPS-mediated MMP9 induction, the role of de novo proteins in MMP9 induction was further investigated. We found that the casedulones inhibited the induction of interleukin-6 (IL-6), a key inflammatory cytokine that participates in STAT3 activation. Moreover, tumor necrosis factor-α (TNFα)-mediated MMP9 induction was significantly suppressed in the presence of the casedulones. Taken together, these findings suggest that casedulones inhibit the IL-6/STAT3 and TNFα pathways, which all involve LPS-mediated MMP9 induction.

Isolation of six isoprenylated biflavonoids from the leaves of Garcinia subelliptica.

The acetone-soluble parts of Garcinia subelliptica leaves were analyzed and six new biflavonoids were isolated, i.e., garciniaflavones A-F (1-6), as well as the five known biflavonoids amentoflavone (7), podocarpusflavone A (8), (+)-morelloflavone (9), (+)-morelloflavone-7"-O-ß-glucopyranoside (10), and (+)-4'''-O-methylmorelloflavone (11) and the three triterpenoids oleanan-3-one, ß-amyrin, and cycloartenol. The structures of the isolates were established based on spectroscopic analyses, including a detailed NMR spectroscopic investigation. The new biflavonoids are rare mono-isoprenylated derivatives that have a flavone-(3'-8")-flavone core (1-4: amentoflavone type) and a flavanone-(3-8")-flavone core (5, 6: morelloflavone type). The absolute configurations of the morelloflavone-type biflavonoids (5, 6) were confirmed by circular dichroism to be 2R,3S. The biflavonoids with an isoprenyloxy group (1) and a 2-hydroxy-3-methyl-3-butenyl group (2), and the morelloflavone-type biflavonoids with a C(5) unit are the first examples in nature. We found that 7, one of the major biflavonoids, strongly inhibited hypoxia-inducible factor-1 in human embryonic kidney 293 cells under hypoxic conditions.

Vitamin E derivative ETS-GS reduces liver ischemia-reperfusion injury in rats.

BACKGROUND: Ischemic liver injury is often the result of surgical procedures such as liver transplantation and hepatic resection. Liver damage occurs after reperfusion, leading to increased systemic inflammation. Recent studies have reported that vitamin E and glutathione can ameliorate ischemia-reperfusion (I/R) injury. In the present study, we evaluated the ability of a new vitamin E derivative, ETS-GS, to improve liver I/R injury. MATERIALS AND METHODS: Male Wistar received a subcutaneous injection of ETS-GS (10 mg/kg) or saline before experimentally-induced liver I/R injury or sham treatment. The rats were sacrificed after the 60-min ischemia and 24-h reperfusion. Histology and serum levels of cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-6, and high-mobility group box 1 (HMGB1) protein] and liver enzymes were determined to evaluate the protective effects of ETS-GS. RESULTS: We found that ETS-GS treatment attenuated I/R-induced histologic alterations, reduced levels of liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). In addition, ETS-GS treatment decreased serum cytokine levels. CONCLUSIONS: Taken together, our results demonstrate that ETS-GS attenuates I/R injury in a rat model and suggests that ETS-GS may exert anti-inflammatory effects. Accordingly, ETS-GS may have therapeutic potential to treat various clinical conditions involving I/R injury.

Terpenoids from Chloranthus serratus and their anti-inflammatory activities.

Seven new terpenoids, including two sesquiterpene dimers (1, 2), two norditerpenoids (3, 4), and three sesquiterpenes (5-7), along with six known sesquiterpene dimers and four known sesquiterpenes were isolated from the whole plant of Chloranthus serratus. Their structures and relative configurations were elucidated on the basis of spectroscopic data analysis. The absolute configuration of 1 was determined by the CD exciton chirality method. These isolates were evaluated for their inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells. Compound 2 and two known compounds, shizukaols B and D, showed significant anti-inflammatory activities, with IC(50) values of 0.22, 0.15, and 7.22 µM, respectively.

Novel zierane- and guaiane-type sesquiterpenes from the root of Melicope denhamii.

Two novel zierane-type sesquiterpenes, named melicodenones A and B (1 and 2, resp.), and three new guaiane-type sesquiterpenes, named melicodenones C-E (3-5), were isolated from the root of Melicope denhamii (Seem.) T. G. Hartley together with zierone (6). Their structures were established by extensive NMR-spectroscopic analyses. Compounds 1-6 were tested for cytotoxicity using human colon cancer DLD-1 cells, and melicodenone A (1) was found to exhibit moderate activity.

Anti-inflammatory sesquiterpenes and sesquiterpene dimers from Chloranthus fortunei.

A novel lindenane sesquiterpene with an unprecedented 18-membered triester ring, named chlorafortulide (1), along with one known lindenane sesquiterpene (2) and nine known lindenane sesquiterpene dimers (3-11), was isolated from the whole plant of Chloranthus fortunei. Their structures and relative configurations were elucidated on the basis of spectroscopic analysis. All the isolates were evaluated for their inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 cells. Henriol D (4), shizukaols E (8), G (9), M (10), and O (11) showed significant anti-inflammatory activities with IC(50) values of 1.90, 3.68, 1.95, 7.01, and 1.95 µM, respectively.

Two new methoxylated flavones isolated from Casimiroa edulis La Llave and their MMP-9 inhibitory activity.

Casimiroa edulis La Llave is known to contain unusual 5,6-dimethoxyflavones bearing a variously oxygenated B-ring. Phytochemical investigation of the leaves and the roots of C. edulis achieved the isolation of two new methoxylated flavones, named casedulones A (1) and B (2), together with 12 known analogues. Their unique structures were established with the aid of spectral analyses and total syntheses. Pre-treatment with 20 µM of 1 and 2 suppressed MMP-9 expression in LPS-mediated THP-1 cells, indicating that the characteristic flavonoids in C. edulis could be potential anti-angiogenics for cancer prevention.

Occurrence of C-glucoside of resveratrol oligomers in Hopea parviflora.

Investigation of the highly polar chemical constituents in the stem of Hopea parviflora (Dipterocarpaceae) resulted in the isolation of four new resveratrol derivatives, hopeasides A and B (1, 2) (resveratrol pentamers), C (3) (resveratrol trimer), and D (4) (resveratrol dimer) together with nine known resveratrol oligomers (5-13). The new structures have a common partial structure of the 1-hydroxy-1-(3,5-dihydroxy-2-C-glucopyranosylphenyl)-2-(4-hydroxyphenyl)ethane-2-yl group after oxidative condensation of (E)-resveratrol-10-C-ß-glucopyranoside (14). The structures were determined by spectroscopic analysis including 2D-NMR and computer-aided molecular modeling. The biogenetic relationship of the isolates and NMR characteristics caused by steric hindrance are also discussed in this paper.

Resveratrol derivatives from Vatica albiramis.

Three new stilbene derivatives, albiraminols A (1) (resveratrol hexamer), B (2) (resveratrol dimer), and vatalbinoside F (3) (mono-glucoside of resveratrol dimer), along with malibatol were isolated from acetone soluble portions of the stem of Vatica albiramis. The structures of the isolates were established on the basis of spectroscopic analyses, including a detailed NMR spectroscopic investigation. The biosynthetic aspects of the isolates are discussed in this paper. Compound 1 is composed of tetrameric resveratrol (vaticanol B (1A)) and dimeric resveratrol (1B) and is the first instance of the resveratrol derivative bearing a 5,6,11,12-tetrahydro-5,11-epoxydibenzo[a,e][8]annulene ring system. Compound 2 possesses a novel 4,5-dihydro-13-oxabenzo[3,4]azuleno[7,8,1-jkl]phenanthrene skeleton in the framework.

Resveratrol oligomers from Vatica albiramis.

Five new stilbenoids, vatalbinosides A-E (1-5), and 13 known compounds (6-18) were isolated from the stem of Vatica albiramis. The effects of these new compounds on interleukin-1ß-induced production of matrix metalloproteinase-1 (MMP-1) in human dermal fibroblasts were examined. Three resveratrol tetramers, (-)-hopeaphenol (6), vaticanol C (13), and stenophyllol C (14), were identified as strong inhibitors of MMP-1 production.

Inhibitory effects of flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki 'Fuyu') on melanin biosynthesis.

We found that the acetone extract of the peel of Japanese persimmon (Diospyros kaki 'Fuyu') inhibits melanin biosynthesis in mouse B16 melanoma cells. The activity-guided purification of the extract resulted in isolation of two active compounds, which have been identified as flavonoid glycosides, isoquercitrin (quercetin-3-O-glucoside) and hyperin (quercetin-3-O-galactoside) by spectral analysis. Isoquercitrin and hyperin strongly inhibited the production of melanin (IC(50): 21.7 and 18.2 microM, respectively). The inhibitory effects were found to be mediated by suppression of tyrosinase expression.

Agarwood induced laxative effects via acetylcholine receptors on loperamide-induced constipation in mice.

Agarwood (Aquilaria sinensis, Aquilaria crasna) is well known as an incense in the oriental region such as Thailand, Taiwan, and Cambodia, and is used as a digestive in traditional medicine. We investigated the laxative effects and mechanism of agarwood leaves extracted with ethanol (EEA-1, Aquilaria sinensis; EEA-2, Aquilaria crasna). EEA-1, EEA-2, the main constituents of EEAs (mangiferin, and genkwanin-5-O-primeveroside), and senna increased the frequency and weight of stools in loperamide-induced constipation model mice. EEA-1 and EEA-2 did not induce diarrhea as a side effect, but senna induced severe diarrhea. EEA-1 and senna increased gastro-intestinal (GI) transit in the model mice. EEA-1, but not senna, also increased the intestinal tension of isolated jejunum and ileum in guinea pigs, and the tension increase was blocked by atropine, a muscarinic receptor antagonist, but not by other inhibitors (granicetron, pyrilamine, or bradykinin-antagonist peptide). Furthermore, the increase in frequency and weight of stools induced by EEA-1 were blocked by pre-administration of atropine in the model mice. These findings indicate that EEAs exerted a laxative effect via acetylcholine receptors in the mouse constipation model.

Chemical constituents in the leaves of Vateria indica.

Comprehensive re-investigation of the chemical constituents in the leaves of Vateria indica (Dipterocarpaceae) resulted in the isolation of a novel resveratrol dimeric dimer having a C(2)-symmetric structure, vateriaphenol F (1), and two new O-glucosides of resveratrol oligomers, vateriosides A (2) (resveratrol dimer) and B (4) (resveratrol tetramer), along with a new natural compound (3) and 33 known compounds including 26 resveratrol derivatives. The absolute structures were elucidated by spectroscopic analysis, including two dimensional NMR and circular dichroism (CD) spectra.

Discovery and SAR of Natural-Product-Inspired RXR Agonists with Heterodimer Selectivity to PPARδ-RXR.

A known natural product, magnaldehyde B, was identified as an agonist of retinoid X receptor (RXR) α. Magnaldehyde B was isolated from Magnolia obovata (Magnoliaceae) and synthesized along with more potent analogs for screening of their RXRα agonistic activities. Structural optimization of magnaldehyde B resulted in the development of a candidate molecule that displayed a 440-fold increase in potency. Receptor-ligand docking simulations indicated that this molecule has the highest affinity with the ligand binding domain of RXRα among the analogs synthesized in this study. Furthermore, the selective activation of the peroxisome proliferator-activated receptor (PPAR) δ-RXR heterodimer with a stronger efficacy compared to those of PPARα-RXR and PPARγ-RXR was achieved in luciferase reporter assays using the PPAR response element driven reporter (PPRE-Luc). The PPARδ activity of the molecule was significantly inhibited by the antagonists of both RXR and PPARδ, whereas the activity of GW501516 was not affected by the RXR antagonist. Furthermore, the molecule exhibited a particularly weak PPARδ agonistic activity in reporter gene assays using the Gal4 hybrid system. The obtained data therefore suggest that the weak PPARδ agonistic activity of the optimized molecule is synergistically enhanced by its own RXR agonistic activity, indicating the potent agonistic activity of the PPARδ-RXR heterodimer.

Inhibitory effects of sesquiterpene lactones isolated from Eupatorium chinense L. on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice.

Sesquiterpene lactones (SQTLs) have been shown to suppress the degranulation as inferred by histamine release in rat basophilic leukemia RBL-2H3 cells. In this study, we isolated the 9 kinds of SQTLs from Eupatorium chinense L. and examined the effects of these SQTLs on the degranulation in RBL-2H3 cells. The chemical structures of two novel compounds (SQTL-3 and 8) were determined. All the SQTLs suppressed the degranulation from Ag-stimulated RBL-2H3 cells. To disclose the inhibitory mechanism of degranulation by SQTLs, we examined the activation of intracellular signaling molecules such as Lyn, Syk, and PLCgammas and intracellular free Ca(2+) concentration ([Ca(2+)]i). None of these SQTLs showed the activation of Syk and PLCgammas. The intracellular free Ca(2+) concentration ([Ca(2+)]i) was elevated by Fc epsilonRI activation, but SQTLs treatment reduced the elevation of [Ca(2+)]i by suppressing Ca(2+) influx. Thus, it was suggested that the suppression of Ag-stimulated degranulation by these SQTLs is mainly due to the decreased Ca(2+) influx. Furthermore, in order to clarify the in vivo effect of SQTL-rich extract, we administered SQTL-rich extract to the type I allergic model mice and measured the passive cutaneous anaphylaxis (PCA) reaction induced by IgE-antigen complex. The SQTLs remarkably suppressed PCA reaction in a dose-dependent manner. Thus, it was suggested that SQTLs would be a candidate as an anti-allergic agent.

Down-regulation of aquaporin 9 gene transcription by 10-hydroxy-2-decenoic acid: A major fatty acid in royal jelly.

10-Hydroxy-trans-2-decenoic acid (10H2DA) is a unique lipid component of royal jelly produced by worker honeybees that exerts insulin-like effects. We herein investigated the effects of 10H2DA on the gene expression of aquaporin 9 (AQP9), which functions as a glycerol transporter in the liver, to clarify whether 10H2DA modulates energy metabolism. 10H2DA suppressed AQP9 gene expression in HepG2 cells by promoting the phosphorylation of Akt and AMP-activated protein kinase (AMPK). This suppression was partially recovered by the treatment of cells with inhibitors for Akt and AMPK. Based on the result showing that leptomycin B partially recovered the suppression of AQP9 gene expression, 10H2DA inhibited the expression of Foxa2, a transcription factor for the AQP9 gene, and also induced its nuclear exclusion. Although 10H2DA up-regulated phosphoenolpyruvate carboxykinase and glucose-6-phosphatase gene expression, this was suppressed through the modulation of Foxa2 by insulin. These results suggest that 10H2DA suppresses AQP9 gene expression through the phosphorylation of Akt and AMPK and down-regulation of Foxa2 expression.

Flavonol glycosides of Rosa multiflora regulates intestinal barrier function through inhibiting claudin expression in differentiated Caco-2 cells.

Eijitsu, the fruits of Rosa multiflora Thunberg, is a traditional Japanese natural medicine and used as purgatives. The active constituents were identified as flavonol glycosides, multiflorin A (MF), and multinoside A (MSA), but mechanism of the purgative action is still unknown. We hypothesized that the flavonol glycosides 1 and 2 may exhibit the purgative actions through modulating intestinal epithelial barrier function. Then, this study aimed to investigate their effects on intestinal epithelial barrier function and possible molecular mechanisms in human intestinal Caco-2 cells. MF and MSA decreased transepithelial electrical resistance and increased paracellular permeability of Caco-2 cell monolayers. Expression of claudins (CLDNs) involved in paracellular permeability of ions and low-molecular substances was significantly decreased by the treatment with MF or MSA. The compounds increased the ratio of N-cadherin/E-cadherin, expression of transforming growth factor-ß and Slug, and phosphorylation level of Smad3, suggesting epithelial-mesenchymal transition activation, and epithelial-mesenchymal transition inhibition by transforming growth factor-ß receptor kinase inhibitors completely recovered the decreased CLDNs expression caused by MF and MSA. Moreover, the increased paracellular permeability and the decreased CLDNs expression by the treatment with MF or MSA for 24 hours recovered to the same extent as the untreated group with the compounds by continuous culture in the growth medium alone for 48 hours. These results suggest that Eijitsu may be effective in preventing or relieving constipation symptoms, unless used chronically.