Dietary polyacetylene falcarinol upregulated intestinal heme oxygenase-1 and modified plasma cytokine profile in late phase lipopolysaccharide-induced acute inflammation in CB57BL/6 mice.

Unlike polyphenols, which are widely available in the diet, polyacetylenes are available only from the Apiaceae family vegetables, including carrot, parsnip, fennel, celery, and many herbs (parsley, lovage, etc). The aim of this study was to investigate the hypothesis that polyacetylene falcarinol (FA) reduces intestinal inflammation and examine its similarity of effect to isothiocyanate R-sulforaphane during the late phase of acute inflammation. To this end, 3-month-old male CB57BL/6 mice were fed twice daily for 1 week with 5 mg/kg of FA, sulforaphane, or vehicle before receiving an intraperitoneal injection of 5 mg/kg endotoxin (lipopolysaccharide [LPS]) to induce modest acute inflammation. The expression of intestinal and hepatic heme oxygenase-1 at the mRNA and protein levels, circulating cytokines, as well as intestinal and mesenteric n-6 and n-3 fatty acid lipid mediators was compared 24 hours after LPS administration to examine its effects on the late phase of inflammation. Intestinal nuclear factor (erythroid-derived 2)-like 2 target enzyme heme oxygenase-1 was upregulated 8.42-fold at the mRNA level and 10.7-fold at the protein level by FA-supplemented diet. However, the FA-supplemented diet produced a unique type-2 plasma cytokine skew after LPS treatment. Plasma cytokines interleukin (IL)-4, IL-13, IL-9, and IL-10 were upregulated, reflecting the cytokine profile of reduced type 1 inflammation. A detailed lipidomic analysis of n-6 and n-3 fatty acid pro- and anti-inflammatory pathways in the mesentery and intestinal mucosa showed that FA diet was more similar to the control groups than to other LPS treated groups. In this study, we demonstrated that FA-supplemented diet produced a unique immunomodulatory effect not observed with sulforaphane in late phases of inflammation. These results support the hypothesis that FA may have role as a dietary immunosuppressant in patients with inflammatory gastrointestinal as well as other inflammatory disorders that may be alleviated by increasing consumption of carrot or other FA-containing food sources.

Panaxynol from Saposhnikovia diviaricata exhibits a hepatoprotective effect against lipopolysaccharide + D-Gal N induced acute liver injury by inhibiting Nf-κB/IκB-α and activating Nrf2/HO-1 signaling pathways.

We investigated the mechanism of action of panaxynol (PAL) extract from the root of Saposhnikovia diviaricata (Turcz.) Schischk for treating acute liver injury caused by lipopolysaccharide (LPS) and D-galactosamine (D-Gal N) in mice. A mouse model of acute liver failure induced by LPS/D-Gal N was established. Mice were divided randomly into three equal groups: control group, LPS/D-Gal N group and PAL group. After seven days of continuous PAL administration, all animals except controls were injected with 50 µg/kg LPS and 800 mg/kg D-Gal N; blood and liver samples were collected after 8 h. Compared to the LPS/D-Gal N group, the levels of catalase, glutathione and superoxide dismutase were increased in the liver of the PAL group. The inflammatory response index indicated that PAL attenuated LPS/D Gal N-induced liver pathological injury and decreased levels of hepatic malondialdehyde, serum alanine aminotransferase, aspartate transaminase, tumor necrosis factor-α, and interleukins 1ß and 6. PAL also inhibited LPS/D-Gal N induced nuclear factor-kappa B (Nf-κB), inhibitor kappa B-α (IκB-α) activation, and up-regulated Nrf2 and heme oxygenase-1 (HO-1) expression. PAL can prevent LPS/D-Gal N induced acute liver injury by activating Nrf2/HO-1 to stimulate antioxidant defense and inhibit the IkB-α/NF-κB signaling pathway.

Dietary Polyacetylenic Oxylipins Falcarinol and Falcarindiol Prevent Inflammation and Colorectal Neoplastic Transformation: A Mechanistic and Dose-Response Study in A Rat Model.

Falcarinol (FaOH) and falcarindiol (FaDOH) are cytotoxic and anti-inflammatory polyacetylenic oxylipins, which are commonly found in the carrot family (Apiaceae). FaOH and FaDOH have previously demonstrated a chemopreventive effect on precursor lesions of colorectal cancer (CRC) in azoxymethane (AOM)-induced rats. The purpose of the present study was to elucidate possible mechanisms of action for the preventive effect of FaOH and FaDOH on colorectal precancerous lesions and to determine how this effect was dependent on dose. Gene expression studies performed by RT-qPCR of selected cancer biomarkers in tissue from biopsies of neoplastic tissue revealed that FaOH and FaDOH downregulated NF-κß and its downstream inflammatory markers TNFα, IL-6, and COX-2. The dose-dependent anti-neoplastic effect of FaOH and FaDOH in AOM-induced rats was investigated in groups of 20 rats receiving a standard rat diet (SRD) supplemented with 0.16, 0.48, 1.4, 7 or 35 µg FaOH and FaDOH g-1 feed in the ratio 1:1 and 20 rats were controls receiving only SRD. Analysis of aberrant crypt foci (ACF) showed that the average number of small ACF (<7 crypts) and large ACF (>7 crypts) decreased with increasing dose of FaOH and FaDOH and that this inhibitory effect on early neoplastic formation of ACF was dose-dependent, which was also the case for the total number of macroscopic neoplasms. The CRC protective effects of apiaceous vegetables are mainly due to the inhibitory effect of FaOH and FaDOH on NF-κB and its downstream inflammatory markers, especially COX-2.

Chemo-Preventive Potential of Falcarindiol-Enriched Fraction from Oplopanax elatus on Colorectal Cancer Interfered by Human Gut Microbiota.

Oplopanax elatus (Nakai) Nakai is an oriental herb, the polyyne-enriched fraction of which (PEFO) showed anticolorectal cancer (anti-CRC) effects. Other concomitant components, which are inevitably bio-transformed by gut microbiota after oral administration, might be interfere with the pharmacodynamics of polyynes. However, the influence of human gut microbiota on molecules from O. elatus possessing anticancer activity are yet unknown. In this study, the compounds in PEFO and PEFO incubated with human gut microbiota were analyzed and tentatively identified by HPLC-DAD-QTOF-MS. Two main polyynes ((3S,8S)-falcarindiol and oplopandiol) were not significantly decomposed, but some new unknown molecules were discovered during incubation. However, the antiproliferative effects of PEFO incubated with human gut microbiota for 72 h (PEFO I) were much lower than that of PEFO on HCT-116, SW-480, and HT-29 cells. Furthermore, PEFO possessed better anti-CRC activity in vivo, and significantly induced apoptosis of the CRC cells, which was associated with activation of caspase-3 according to the Western-blot results (P<0.05). These results suggest anticolorectal cancer activity of polyynes might be antagonized by some bio-converted metabolites after incubation with human gut microbiota. Therefore, it might be better for CRC prevention if the polyynes could be orally administrated as purified compounds.

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 ± 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

Dietary polyacetylenes, falcarinol and falcarindiol, isolated from carrots prevents the formation of neoplastic lesions in the colon of azoxymethane-induced rats.

Falcarinol (FaOH) and falcarindiol (FaDOH) are found in many food plants of the Apiaceae family. Carrots are a major dietary source of these polyacetylenes. Feeding azoxymethane (AOM)-induced rats with carrots and purified FaOH have previously been shown to inhibit neoplastic transformations in the colon. FaOH and FaDOH have also shown to have a synergistic effect in vitro, resulting in a significant increased cytotoxic activity. Based on these findings the antineoplastic effect of FaOH and FaDOH (purity > 99%) was investigated in the AOM-induced rat model. Twenty rats received rat diet containing 7 µg FaOH per g feed and 7 µg FaDOH per g feed and 20 rats were controls receiving only rat diet. Then carcinogenesis was induced in all 40 rats with the carcinogen AOM. All animals received the designated diet for 2 weeks before AOM induction and continued on the designated diet throughout the experiment. Rats were euthanized 18 weeks after the first AOM injection and macroscopic polyp/cancers were measured, harvested and stained for histology. The difference in sizes of aberrant crypt foci (ACF) were analysed in a Wilcoxon rank sum test, in which the median number of small ACF was 218 in controls and 145 in polyacetylene treated rats (P < 0.001). Fifteen control rats and 8 treated rats had macroscopic tumors (P = 0.027). The number of tumors larger than 3 mm were 6 and 1 in control and treated rats, respectively (P = 0.032). In conclusion dietary supplements with FaOH and FaDOH reduced the number of neoplastic lesions as well as the growth rate of the polyps suggesting a preventive effect of FaOH and FaDOH on the development of colorectal cancer.

Pharmacokinetic profiles of falcarindiol and oplopandiol in rats after oral administration of polyynes extract of Oplopanax elatus.

Polyynes, such as facarindiol (FAD) and oplopandiol (OPD), are responsible for anticancer activities of Oplopanax elatus (O. elatus). A novel approach to pharmacokinetics determination of the two natural polyynes in rats was developed and validated using a liquid chromatography-electrospray ionization-mass spectrometry (LC-MS) method. Biosamples were prepared by liquid-liquid extraction using ethyl acetate/n-hexane (V : V = 9 : 1) and the analytes were eluted on an Agilent ZORBAX Eclipse Plus C18 threaded column (4.6 mm × 50 mm, 1.8 µm) with the mobile phase of acetonitrile-0.1% aqueous formic acid at a flow-rate of 0.5 mL·min(-1) within a total run time of 11 min. All analytes were simultaneously monitored in a single-quadrupole mass spectrometer in the selected ion monitoring (SIM) mode using electrospray source in positive mode. The method was demonstrated to be rapid, sensitive, and reliable, and it was successfully applied to the pharmacokinetic studies of the two polyynes in rat plasma after oral administration of polyynes extract of O. elatus.

Inhibition of glycogen synthase kinase-3ß by falcarindiol isolated from Japanese Parsley (Oenanthe javanica).

A new biological activity of falcarindiol isolated from Japanese parsley (Oenanthe javanica) using the mutant yeast YNS17 strain (zds1Δ erg3Δ pdr1Δ pdr3Δ) was discovered as an inhibitor of glycogen synthase kinase-3ß (GSK-3ß). Falcarindiol inhibited GSK-3ß in an ATP noncompetitive manner with a Ki value of 86.9 µM using a human enzyme and luminescent kinase assay platform. Falcarindiol also both suppressed gene expression of glucose-6-phosphatase (G6Pase) in rat hepatoma H4IIE cells and protected mouse neuroblastoma HT22 cells from glutamate-induced oxidative cell death at 10 µM. During an oral glucose tolerance test (OGTT), the blood glucose level was significantly decreased in the rats treated with oral administration of O. javanica extract containing falcarindiol (15 mg/kg). These findings indicate that Japanese parsley could be a useful food ingredient against type-2 diabetes and Alzheimer's disease.

The herbal medicine compound falcarindiol from Notopterygii Rhizoma suppresses dendritic cell maturation.

Dendritic cells (DCs) are important for regulating the immune response. We report an herbal medicine compound called falcarindiol that affects DC function. Ethanol extracts of 99 crude drugs that are the main components of 210 traditional Japanese medicines (Kampo medicine) approved by the Ministry of Health, Labor and Welfare in Japan were prepared and screened using the murine epidermal-derived Langerhans cell line XS106. Notopterygii Rhizoma strongly suppressed major histocompatibility complex (MHC) class II expression in XS106 cells. Activity-guided fractionation led to the isolation and identification of falcarindiol as a principal active compound in Notopterygii Rhizoma. Falcarindiol (1-5 microM) dose-dependently suppressed MHC II expression in XS106 cells. Fresh-isolated bone marrow-derived DCs were examined for the production of MHC II, CD80, CD86, interleukin (IL)-12p70, and IL-10. Treatment of bone marrow-derived DCs with 5 muM falcarindiol significantly inhibited lipopolysaccharide-induced phenotype activation and cytokine secretion and inhibited MHC II expression by CD40 ligation, but not phorbol 12-myristate 13-acetate + ionomycin or IL-12. Falcarindiol inhibited DC maturation by blocking the canonical pathway of nuclear factor-kappaB and phosphorylated p38. Topical application of 0.002 and 0.01% falcarindiol before sensitization dose-dependently suppressed delayed-type hypersensitivity to ovalbumin (p < 0.01). Falcarindiol induces immunosuppressive effects in vitro and in vivo and might be a novel therapy for autoimmune or allergic diseases.

Differential effects of falcarinol and related aliphatic C(17)-polyacetylenes on intestinal cell proliferation.

Quantitative major polyacetylenes of carrots (falcarinol and falcarindiol) and American ginseng roots (falcarinol and panaxydol) were isolated and tested in human intestinal epithelial cells of normal (FHs 74 Int.) and cancer (Caco-2) origin. A hormesis effect was seen for all isolated polyacetylenes when added to Caco-2 cells in concentrations ranging from 1 ng/mL to 20 microg/mL. The relative inhibitory potency was falcarinol > panaxydol > falcarindiol. No hormesis effect was observed when adding the polyacetylenes to FHs 74 Int. cells. Instead, an inhibitory growth response was observed above 1 microg/mL. The relative inhibitory potency was panaxydol > falcarinol > falcarindiol. Maximal inhibition at 20 microg/mL corresponded to approximately 95% and 80% inhibition of cell proliferation in normal and cancer cells, respectively. Combinations of falcarinol and falcarindiol added to normal and cancer cells showed a synergistic response for the inhibition of cell growth. Furthermore, the oxidized form of falcarinol, falcarinon, showed a significantly less growth inhibitory effect in intestinal cells of both normal and cancer origin; hence, a hydroxyl group at C-3 may be important for activity of falcarinol-type polyacetylenes. Extracts of carrots, containing different amounts of falcarinol, falcarindiol, and falcarindiol 3-acetate had significant inhibitory effects on both normal and cancer cell proliferation. In cancer cells, the extract containing the highest concentration of falcarinol tended to have the highest growth inhibitory effect, in accordance with a higher potency of falcarinol than falcarindiol. The present study demonstrates that aliphatic C(17)-polyacetylenes are potential anticancer principles of carrots and related vegetables and that synergistic interaction between bioactive polyacetylenes may be important for their bioactivity.

The polyacetylene falcarindiol with COX-1 activity isolated from Aegopodium podagraria L.

Extracts of Aegopodium podagraria L. were screened in vitro for cyclooxygenase-1 (COX-1) inhibitory activity. The isolation of the active compound falcarindiol was achieved by bioassay-guided fractionation. The identification of the active compound was confirmed by (1)H NMR and (13)C NMR. The IC(50)-value of falcarindiol was 0.3 microM in the COX-1 assay. A quantitative determination of the seasonal variation in the content of falcarindiol in different plant parts was carried out by HPLC analysis. The flowers from Aegopodium podagraria collected in June 2006 had the highest concentration of falcarindiol (88 mg/g plant material).

A small molecule Smac mimic potentiates TRAIL-mediated cell death of ovarian cancer cells.

OBJECTIVES: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (Smac) has been described to sensitize for apoptosis. We have explored the proapoptotic activity of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines (A2780 cells and its chemoresistant derivatives A2780/ADR and A2780/DDP), cancer cell lines and in primary ovarian cancer cells. METHODS: The effects of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines and primary ovarian cancer cells were determined by cell proliferation, apoptosis and biochemical assays. RESULTS: This compound added alone elicited only a weak proapoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAILR2 antibody (Lexatumumab) in inducing apoptosis of ovarian cancer cells. CONCLUSIONS: These observations suggest that small molecule mimic of Smac/DIABLO could be useful for the development of experimental strategies aiming to treat ovarian cancer. Interestingly, in addition to its well known proapoptotic effects, Smac/DIABLO elicited a significant increase of pro-caspase-3 levels.