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.

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.

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.

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.

Pteleifolols A-E, acetophenone di-C-glycosides and a benzopyran dimer from the leaves of Melicope pteleifolia.

Four new acetophenone di-C-glycosides, pteleifolols A-D (1-4) and a new dimeric benzopyran, pteleifolol E (5), were isolated from the leaves of Melicope pteleifolia. Seven known compounds, including 2,4,6-trihydroxyacetophenone-3,5-di-C-glucopyranoside (6), were also isolated. Structures of the new compounds (1-5) were established by using spectroscopic and spectrometric techniques, including 1D and 2D nuclear magnetic resonance (NMR), UV, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data. Pteleifolols A-D (1-4) were E-p-coumaroyl, Z-p-coumaroyl, E-feruloyl, and benzoyl esters of 6, respectively. Pteleifolol E (5) was a dichromene dimerized through a C2 unit.

Yuccalides A-C, three new phenolic compounds with spiro-structures from the roots of Yucca gloriosa.

Three new phenolic compounds, yuccalides A-C (1-3), were isolated from the roots of Yucca gloriosa L., along with four known compounds (4-7). The structures of the new compounds were established by extensive NMR spectroscopic analyses. Inducible nitric oxide synthase (iNOS) mRNA levels induced by lipopolysaccharide (LPS) in mouse macrophage-like RAW 264.7 cells were effectively suppressed by compounds 2, 4, and 6, all of which had the (2R*, 3R*)-configuration. IL-1ß and IL-6 mRNA levels induced by LPS were significantly attenuated by compounds 4, 5, and 6, but not by 2.

A New Flavonol Triglycoside from Eustoma grandiflorum.

A new flavonol triglycoside, kaempferol 3-0-α-L-rhamnopyranosyl (1>6)-(3-0-E-p-coumaroyl)- ß-D-galactopyranoside-7-0-α-L-rhamnopyranoside (1: Eustograndifloside A) was isolated from the flower of Eustoma grandiflonm in addition to eight known flavonols (2: kaempferol 3-0-a-L-rhamnopyranosyl (1->6)-(4-0-E-p-coumdarhyl)-m-D-galactopyraoside-7-0-α-L-rhamnopyranoside, ,3: kaempferol 3-0-ß-robinobioside-7-0-α-L-rhamnopyranoside, 4: isorhamne-tin 3-0-α-L-rhamnopyranosyl (1->2). [α-L-rhamnopyranosyl (1->6)]-(4-O-E-p-couinaroyl)-ß-D-galactopyrAnoside-7-0-α-L-rhamnopyranoside, 5: kaempferol 3-0-α-L-rhamnopyranosyl (1->2) [(X-L-rhamnopyranosyl (1-6)] (4-0-E-p-coumaroyl)-ß-D-galactopyranoside-7-0-α-L-rhamnopyranoside, 6: kaempferol 3-0-ß-robinobioside, 7: quercetin 3-0-ß-robinobioside, 8: isorhamnetin 3-0-ß-robinobioside, and 9: kaempferol 7-0-α-L-rhamnopyranoside) and two known secoiridoid glycosides (10: swertiamarin and 11: sweroside). The structure elucidation of these compounds was accomplished through analyses of spectroscopic, including 1D and 2D NMR, and ESIMS data.

Anti-Androgenic Activity of Icarisid II from Epimedium Herb in Prostate Cancer LNCaP Cells.

Anti-androgens are regarded as potential therapeutic agents for the treatment of prostate cancer. We determined that an epimedium herb (EH) extract exhibited anti-androgenic activity in a luciferase assay using androgen receptor-positive prostate cancer LNCaP cells. Nine EH-derived flavonoids were examined. The results identified icarisid II as a very potent anti-androgenic EH-derived flavonoid. A quantitative RT-PCR analysis confirmed that the flavonol suppressed the expression of the androgen-responsive KLK3 gene.

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.

Structure-activity relationship of flavonoids as potent inhibitors of carbonyl reductase 1 (CBR1).

Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase superfamily, reduces a variety of carbonyl compounds including therapeutic drugs. CBR1 is involved in the reduction of the anthracycline anticancer drugs to their less anticancer C-13 hydroxy metabolites, which are cardiotoxic. CBR1 inhibitors are thought to be promising agents for adjuvant therapy with twofold beneficial effect in prolonging the anticancer efficacy of the anthracyclines while decreasing cardiotoxicity, a side effect of the drugs. In this study, we evaluated 27 flavonoids for their inhibitory activities of CBR1 in order to explore the structure-activity relationship (SAR). Among them, luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one) showed the most potent inhibition (IC5095nM), which is also more potent compared to all known classes of CBR1 inhibitors. The inhibition of luteolin was noncompetitive with respect to the substrate in the NADPH-dependent reduction direction, but CBR1 exhibited moderate NADP(+)-dependent dehydrogenase activity for some alicyclic alcohols, in which the luteolin inhibition was competitive with respect to the alcohol substrate (Ki59nM). The SAR of the flavonoids indicated that the 7-hydroxy group of luteolin was responsible for the potent inhibition of CBR1. The molecular docking of luteolin in CBR1-NADPH complex showed that theflavonoid binds to the substrate-binding cleft, in which its 7-hydroxy group formed a H-bond with main-chain oxygen of Met234, in addition to H-bond interactions (of its 5-hydroxy and 4-carbonyl groups with catalytically important residues Tyr193 and/or Ser139) and a π-stacking interaction (between its phenyl ring and Trp229).

Anti-androgenic activity of hydroxyxanthones in prostate cancer LNCaP cells.

Anti-androgens are used to treat prostate cancer. Here, we report that hydroxyxanthones from a plant extract act as anti-androgens in androgen receptor (AR)-positive prostate cancer LNCaP cells. Anti-androgenic activity of the ethanol extract from Garcinia subelliptica was observed in a luciferase assay using LNCaP/MMTV cells with a stably integrated mouse mammary tumor virus (MMTV) promoter. HPLC-based activity profiling followed by a chemical library-based assay strategy enabled the rapid identification of several active principles bearing a xanthone core substituted with hydroxyl and isoprenyl groups. Among the active compounds, 2-(1,1-dimethyl-allyl)-1,4,5,6-tetrahydroxyxanthone (subelliptenone F) was identified as a potent inhibitor of AR transcriptional activity. The structure-activity relationship of some substituents on the xanthone core was also determined using the chemical library-based bioassay. A quantitative RT-PCR analysis revealed that treatment with the compound resulted in a significant reduction in AR-induced gene (KLK3) expression. Hydroxyxanthone may be a possible candidate for the development of a new anti-androgenic molecule.

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.

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.

Sesquiterpenes from the aerial part of Chloranthus japonicus and their cytotoxicities.

Four new sesquiterpenes, chlorajapolides F-I (1-4), along with nine known terpenoids (5-13) were isolated from the aerial part of Chloranthus japonicus. Their structures were elucidated on the basis of spectroscopic analysis, and a lindenane sesquiterpene, named 9-hydroxy-heterogorgiolide, previously isolated from the C. japonicus, was revised as its 8-epimer (1a). Moreover, methanol extract (ME), EtOAc fraction (EF), water fraction (WF), and all isolates (1a, 1-13) were evaluated for their cytotoxicities using two human cancer cell lines.

Quantification of polyphenols and pharmacological analysis of water and ethanol-based extracts of cultivated agarwood leaves.

Mangiferin (3) and genkwanin 5-O-ß-primeveroside (5) are the two major bioactive polyphenols with laxative property present in the extracts of agarwood (Aquilaria sinensis) leaves (AL). Here we developed an HPLC method to determine these bioactive components and four other major polyphenols in AL extracts and evaluated the pharmacological equivalence of organic and water extracts. Using mobile phase gradient conditions combined with UV detection at 330 nm, all six compounds were separated and we determined the relative extraction ratios of the six compounds present in A. sinensis extracts that were prepared under different conditions and compared the contents of the two laxative polyphenols present in the 60% ethanol extracts of A. sinensis and A. crassna. The polyphenols present in water extracts of 13 commercially cultivated A. crassna plants have also been analyzed. The laxative properties of 60% ethanol and four water extracts of A. crassna were evaluated by the frequency and weight of stools in loperamide-induced constipation model mice. The pharmacological equivalence of 60% ethanol extract and hot water (95°C) extract was identified in mice.

Antiandrogenic activity of resveratrol analogs in prostate cancer LNCaP cells.

The suppression of androgen signaling is a therapeutic target for the treatment of prostate cancer. Resveratrol (3,4',5-trihydroxystilbene) is known to inhibit the function of the androgen receptor (AR). In the present study, we investigated the antiandrogenic activities of resveratrol analogs in order to identify a potent antiandrogen compound. Resveratrol analogs were isolated from plants or were semisynthesized from resveratrol. AR transcriptional activity was measured in prostate cancer LNCaP cells using a luciferase assay with the MMTV-luc reporter plasmid. Among the resveratrol analogs tested, 4'-O-methylresveratrol (3,5-dihydroxy-4'-methoxystilbene) was the most effective inhibitor of AR transcriptional activity. Introduction of a methoxy group to the C-4' of resveratrol and its analogs increased their antiandrogenic activity compared with the unmodified counterparts. Conversely, modification of the 3- and/or 5-hydroxyl groups reduced the antiandrogenic activity. 4'-O-methylresveratrol was more effective than resveratrol in inhibiting Akt phosphorylation, which is related to AR signaling, in LNCaP cells. The hydroxyl groups in resveratrol play a key role in their antiandrogenic effect by modulating AR transcriptional 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.

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.