Dieckol isolated from a brown alga, Eisenia nipponica, suppresses ear swelling from allergic inflammation in mouse.

We previously found a lipophilic fraction of the methanol/chloroform extract of a brown alga, Eisenia nipponica, that had an antiallergic effect in a murine ear swelling test. In this study, we purified the active component from the lipophilic fraction using high performance liquid chromatography and analyzed the mass and nuclear magnetic resonance spectra. This uncovered the phlorotannin dieckol, which exhibited antiallergic effects in an ear swelling test using mice sensitized by arachidonic acid, 12-O-tetradecanoylphorbol-13-acetate, and oxazolone. Mechanistic investigations indicated that dieckol suppressed degranulation, chemical mediator release, and the expression of mRNA such as cyclooxygenase-2, interleukin-6, and tumor necrosis factor-α in rat basophilic leukemia-2H3 cells. In summary, we isolated dieckol from E. nipponica and demonstrated its antiallergic mechanisms. PRACTICAL APPLICATIONS: As the incidence of allergies increases worldwide, so too does the demand for food components with antiallergic and anti-inflammatory properties. Given this trend, we focused on a brown alga that displays a variety of bioactivities. Here, we have isolated dieckol from the antiallergic lipophilic fraction of E. nipponica and found that it possesses diverse physiological activities that may prevent lifestyle-related diseases. Consequently, dieckol or the alga containing this phlorotannin could be used as a health food ingredient to combat not only allergies, but also variety of disorders including the undesirable effects of aging.

Orally Administered Phlorotannins from Eisenia arborea Suppress Chemical Mediator Release and Cyclooxygenase-2 Signaling to Alleviate Mouse Ear Swelling.

Phlorotannin is the collective term for polyphenols derived from brown algae belonging to the genera Ascopyllum, Ecklonia, Eisenia, Fucus and Sargassum etc. Since the incidence of allergies is currently increasing in the world, there is a focus on phlorotannins having anti-allergic and anti-inflammatory effects. In this study, six purified phlorotannins (eckol; 6,6'-bieckol; 6,8'-bieckol; 8,8'-bieckol; phlorofucofuroeckol (PFF)-A and PFF-B) from Eisenia arborea, orally administered to mice, were examined for their suppression effects on ear swelling. In considering the suppression, we also examined whether the phlorotannins suppressed release of chemical mediators (histamine, leukotriene B4 and prostaglandin E2), and mRNA expression and/or the activity of cyclooxygenase-2 (COX-2), using RBL-2H3 cells, a cultured mast cell model. Results showed that the phlorotnannins exhibited suppression effects in all experiments, with 6,8'-bieckol, 8,8'-bieckol and PFF-A showing the strongest of these effects. In conclusion, orally administered phlorotannins suppress mouse ear swelling, and this mechanism apparently involves suppression of chemical mediator release and COX-2 mRNA expression or activity. This is the first report of the anti-allergic effects of the orally administered purified phlorotannins in vivo. Phlorotannins show potential for use in functional foods or drugs.

Immunoreactive intensity of FXPRL amide neuropeptides in response to environmental conditions in the silkworm, Bombyx mori.

In the silkworm Bombyx mori, the diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is a neuropeptide gene that encodes a polypeptide precursor consisting in five Phe-X-Pro-Arg-Leu-NH(2) (FXPRL) amide (FXPRLa) neuropeptides; DH (diapause hormone), PBAN (pheromone-biosynthesis-activating neuropeptide) and α-, ß- and γ-SGNPs (subesophageal ganglion neuropeptides). These neuropeptides are synthesized in DH-PBAN-producing neurosecretory cells contained within three neuromeres, four mandibular cells, six maxillary cells, two labial cells (SLb) and four lateral cells of the subesophageal ganglion. DH is solely responsible, among the FXPRLa peptide family, for embryonic diapause. Functional differentiation has been previously suggested to occur at each neuromere, with the SLb cells releasing DH through brain innervation in order to induce embryonic diapause. We have investigated the immunoreactive intensity of DH in the SLb when thermal (25°C or 15°C) and light (continuous illumination or darkness) conditions are altered and following brain surgery that induces diapause or non-diapause eggs in the progeny. We have also examined the immunoreactivity of the other FXPRLa peptides by using anti-ß-SGNP and anti-PBAN antibodies. Pupal SLb somata immunoreactivities seem to be affected by both thermal and light conditions during embryogenesis. Thus, we have been able to identify a close correlation between the immunoreactive intensity of neuropeptides and environmental conditions relating to the determination of embryonic diapause in B. mori.

Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause.

Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8 kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8 kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8 kDa proteins function to block permeability of O(2) during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8 kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis.

Structure-activity relationship of a novel pentapeptide with cancer cell growth-inhibitory activity.

We previously reported that yamamarin, a pentapeptide with an amidated C-terminus (DILRG-NH(2)) isolated from larvae of the silkmoth, and its palmitoylated analog (C16-DILRG-NH(2)) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure-activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C-terminal part (-RG-NH(2)) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG-NH(2) and C16-DILRG-NH(2) revealed that the N-terminal palmitoyl group of C16-DILRG-NH(2) did not affect the conformation of the C-terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs.

A palmitoyl conjugate of insect pentapeptide Yamamarin arrests cell proliferation and respiration.

A palmitoyl conjugate of an insect pentapeptide that occurs in diapausing insects causes a reversible cell-cycle arrest and suppresses mitochondrial respiration. This peptide compound also causes growth arrest in murine leukemic cell line expressing human gene Bcr/Abl and a farnesoyl peptide induces embryonic diapause in Bombyx mori. These results demonstrate that the insect peptide compounds can lead to the understanding of a common pathway in developmental arrest in animals and may provide a new peptidominetic analog in the development of biopharmaceuticals and pest management.

FXPRL-amide peptides induce ecdysteroidogenesis through a G-protein coupled receptor expressed in the prothoracic gland of Bombyx mori.

The FXPRL-amide peptide family (pyrokinin/PBAN family) consists of insect peptides that function broadly in insect life processes and are characterized by a conserved C-terminal motif. In the silkworm, Bombyx mori, sex pheromone biosynthesis and induction of embryonic diapause are regulated by peptides from this family. To elucidate other functions of Bombyx FXPRL-amide peptides, we analyzed the tissue expression patterns of two known Bombyx G-protein coupled receptors for these peptides. We found that the Bombyx diapause hormone receptor (BmDHR), is expressed in the prothoracic gland (PG), the organ which synthesizes and releases the insect molting hormones, ecdysteroids. Furthermore, diapause hormone (DH), a member of the Bombyx FXPRL-amide peptides, increases both intracellular Ca(2+) and cAMP concentrations and induces ecdysteroidogenesis in late fifth instar PGs coincident with BmDHR expression in the PGs. DH also has the highest prothoracicotropic activity among the FXPRL-amide peptides, which corresponds well to the ligand specificity of heterologously expressed BmDHR. These results demonstrate that FXPRL-amide peptides can function as prothoracicotropic factors through the activation of BmDHR and may play an important role in controlling molting and metamorphosis.

Thermodynamic effects of disulfide bond on thermal unfolding of the starch-binding domain of Aspergillus niger glucoamylase.

The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with beta-cyclodextrin at 25 degrees C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t1/2 of the two mutant proteins decreased by about 10 degrees C as compared to the wild-type protein at pH 7.0. At t1/2 of the wild-type protein (52.7 degrees C), the mutant proteins destabilized by about 10 kJ mol(-1) in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.

Isolation of a new anti-allergic phlorotannin, phlorofucofuroeckol-B, from an edible brown alga, Eisenia arborea.

Eisenia arborea is an edible brown alga occasionally used as a folk medicine in gynecopathy in Japan. A new phlorotannin was isolated from the alga during our search for naturally occurring anti-allergic compounds from edible algae guided by the inhibitory effect on histamine release from rat basophile leukemia (RBL)-2H3 cells. The phlorotannin was called "phlorofucofuroeckol-B." Its structure was determined by spectral analysis and chemical conversion. This paper describes the isolation, structure elucidation, and inhibitory effect of phlorofucofuroeckol-B on histamine release.

G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause.

Bombyx diapause hormone was the first chemical substance identified as a maternal control factor that arrests offspring development. However, the molecular mechanisms by which the hormone transduces the signal to the oocyte that induces embryonic diapause immediately after mesoderm segmentation are not fully understood. Here, we describe a cDNA for a G protein-coupled diapause hormone receptor with seven transmembrane domains. Its amino-acid sequence shows a high level of similarity to the receptors of mammalian neuromedin U and insect regulatory peptide, an FXPRL-amide C-terminus. When expressed in a Xenopus oocyte system, the receptor exhibited the highest affinity (EC(50), approximately 70nM) for diapause hormone, when compared with other Bombyx FXPR/KL-amide peptides. Diapause hormone without amidation at the C-terminus, which never induces embryonic diapause in vivo, had no effect in this heterologous expression system. The mRNA is expressed in the ovaries during Bombyx pupal-adult development. These results strongly indicate that the cDNA encodes the diapause hormone receptor.

Isolation of five types of flavonol from seabuckthorn (Hippophae rhamnoides) and induction of apoptosis by some of the flavonols in human promyelotic leukemia HL-60 cells.

Five types of flavonol were isolated from seabuckthorn (Hippophae rhamnoides) and identified by mass, 1H- and 13C-NMR. The proliferations of human promyelotic leukemia HL-60 cells were inhibited as the concentrations of these flavonols were increased. The order of the extent of growth inhibition by the flavonols at a concentration of 20 microM is as follows: pentamethylquercetin > syringetin > isorhamnetin > quercetin > kaempherol > myricetin. Apoptotic morphological changes of the nucleus, including chromatin condensation were induced in the HL-60 cells treated with quercetin, kaempherol and myricetin, respectively, but not in the cells treated with the other flavonols. The fragmentations of DNA by quercetin, kaempherol and myricetin, respectively, to oligonucleosomal-sized fragments, a characteristic of apoptosis, were observed to be dose-dependent in the HL-60 cells. These findings suggest that growth inhibition by quercetin, kaempherol and myricetin, respectively, results from the induction of apoptosis by these flavonols. The other flavonols (pentamethylquercetin, syringetin and isorhamnetin) having methoxy (-OCH3) group inhibited more strongly than the above 3 flavonols without induction of apoptosis in the HL-60 cells. These findings suggest that mechanisms of growth inhibition by pentamethylquercetin, syringetin and isorhamnetin are different from the apoptosis caused by quercetin, kaempherol and myricetin.

Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori.

In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

2-O-methylisohemigossylic acid lactone, a sesquiterpene, isolated from roots of mokumen (Gossampinus malabarica) induces cell death and morphological change indicative of apoptotic chromatin condensation in human promyelotic leukemia HL-60 cells.

2-O-methylisohemigossylic acid lactone, a sesquiterpene, was purified from roots of mokumen (Gossampinus malabarica) and identified by Mass, and (1)H- and (13)-NMR. This sesquiterpene displayed strong growth inhibitory effect against human promyelotic leukemia HL-60 cells. Apoptotic morphological change of the nucleus, including chromatin condensation was induced in the HL-60 cells treated with the sesquiterpene. The fragmentation of DNA by the sesquiterpene to oligonucleosomal-sized fragments, a characteristic of apoptosis, was observed to be dose- and time-dependent in the HL-60 cells. Inhibitors of caspases suppressed the DNA fragmentation induced by the sesquiterpene. These findings suggest that growth inhibition by the sesquiterpene of HL-60 cells results from the induction of apoptosis by the sesqui-terpene, and that caspase cascade is involved in the induction of apoptosis by the compound in the HL-60 cells.

Induction of apoptosis by maackiain and trifolirhizin (maackiain glycoside) isolated from sanzukon (Sophora Subprostrate Chen et T. Chen) in human promyelotic leukemia HL-60 cells.

We have investigated the effects of maackiain and trifolirhizin (maackiain glycoside) isolated from sanzukon (Sophora Subprostrate Chen et T. Chen) on DNA of human promyelotic HL-60 leukemia cells. It was found that extent of induction of apoptosis by maackiain was larger than that by trifolirhizin in human leukemia HL-60 cells. Morphological changes showing apoptotic bodies were observed in the HL-60 cells treated with maackiain and trifolirhizin. The fragmentations of DNA by maackiain and trifolirhizin to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in the HL-60 cells. The data of the present study show that the suppressions by maackiain and trifolrhizin of growth of the HL-60 cells result from the induction of apoptosis by these compounds, and that the extent of growth suppression and induction of apoptosis by maackiain was greater than that by the glycoside (trifolirhizin).

Induction of apoptosis by lupeol isolated from mokumen (Gossampinus malabarica L. Merr) in human promyelotic leukemia HL-60 cells.

We have investigated the effect of lupeol (lup-20(29)-ene-3beta-ene-3-ol) isolated from mokumen (Gossampinus malabarica L. Merr) on DNA of human promyelotic HL-60 leukemia cells. Induction of apoptosis by lupeol was observed in human leukemia HL-60 cells. Morphological change showing apoptotic bodies was observed in the HL-60 cells treated with lupeol. The fragmentation of DNA by lupeol to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in the HL-60 cells. Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 28.5, 42.0 and 70.9% after a 3-day treatment with 75, 100 and 150 micro M lupeol, respectively. The data of the present study show that the suppression by lupeol of growth of the HL-60 cells results from the induction of apoptosis by this compound.

Hot water soluble sesquiterpenes [anhydroperoxy-costunolide and 3-oxoeudesma-1,4(15),11(13)triene-12,6alpha-olide] isolated from laurel (Laurus nobilis L.) induce cell death and morphological change indicative of apoptotic chromatin condensation in leukemia cells.

Hot water soluble (HWS)-sesquiterpenes [anhydroperoxycostunolide and 3-oxo-eudesma-1,4(15),11(13)triene-12,6alpha-olide] were purified from Laurel (Laurus nobilis L.) and identified by Mass, and 1H- and 13C-NMR. These HWS-sesquiterpenes displayed strong growth inhibitory effect against human promyelotic leukemia HL-60 cells. Apoptotic morphological changes of the nucleus, including chromatin condensation were induced in the HL-60 cells treated with the sesquiterpenes. Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 10.5, 46.5, and 91.3% after a 3 day-treatment with 2.5, 5 and 10 micromoles anhydroperoxycostunolide, respectively. And the same analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 9.8, 39.2 and 89.6% after a 3 day-treatment with 5, 10 and 20 micromoles 3-oxo-eudesma-1,4(15),11(13)triene-12,6alpha-olide, respectively. These findings suggest that growth inhibition by anhydroperoxycostunolide and 3-oxo-eudesma-1,4(15),11(13)triene-12,6alpha-olide of HL-60 cells results from the induction of chromatin condensation in the HL-60 cells. On the other hand, fragmentations by these compounds of DNA to oligonucleosomal-sized fragments were not observed in the HL-60 cells.

Chemistry and antioxidative activity of hot water extract of Japanese radish (daikon).

The comparison of antioxidative activity between the hot water extract and ambient water extract of Japanese radish (daikon) was carried out. The activity of the hot water extract was higher than that of the ambient water extract. One of the antioxidants was isolated as L-tryptophan that should no change in its amount that was determined between the hot water extract and ambient water extract. Moreover, tryptophan changed to 5-hydroxy tryptophan in the rat liver microsome model. This phenomenon may show that tryptophan is changed to another antioxidant in the body.

Sesquiterpenes (costunolide and zaluzanin D) isolated from laurel (Laurus nobilis L.) induce cell death and morphological change indicative of apoptotic chromatin condensation in leukemia HL-60 cells.

Sesquiterpenes (costunolide and zaluzanin D) were purified from laurel (Laurus nobilis L.) and identified by Mass, and 1H- and 13C-NMR. These sesquiterpenes displayed strong growth inhibitory effect against human promyelotic leukemia HL-60 cells. Apoptotic morphological changes of the nucleus, including chromatin condensation were induced in the HL-60 cells treated with the sesquiterpenes. Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 11.4, 47.0, and 92.5% after a 3-day-treatment with 5, 10 and 15 micro M costunolide, respectively. The same analysis showed that the hypodiploid nuclei of HL 60 cells were increased to 12.4, 28.9 and 76.7% after a 3-day-treatment with 10, 15 and 20 micro M zaluzanin D, respectively. These findings suggest that growth inhibition by costunolide and zaluzanin D of HL-60 cells results from the induction of chromatin condensation in the HL-60 cells. On the other hand, fragmentations by these compounds of DNA to oligonucleosomal-sized fragments were not observed in the HL-60 cells.

Cyanidin 3-O-beta-D-glucoside isolated from skin of black Glycine max and other anthocyanins isolated from skin of red grape induce apoptosis in human lymphoid leukemia Molt 4B cells.

Cyanidin 3-O-beta-D-glucoside (CG) was purified from black bean seed coat and other anthocyanins were prepared from red grape skin. These anthocyanins were identified by Mass, and 1H- and 13C-NMR. The effects of four anthocyanins on cell viability in human leukemia Molt 4B cells were investigated. The anthocyanins displayed strong growth inhibitory effects against human leukemia Molt 4B cells. Morphological changes showing apoptotic bodies were observed in the Molt 4B cells treated with these anthocyanins. The fragmentations by these anthocyanins of DNA to oligonucleosomal-sized fragments, that is a characteristic of apoptosis, were observed to be concentration-dependent. N-acetyl-L-cysteine, an antioxidant, suppressed the fragmentation of DNA by these anthocyanins. These findings suggest that growth inhibition of Molt 4B cells by these anthocyanins result from the induction of apoptosis by these compounds and that active oxygen is involved in the induction of apoptosis in the Molt 4B cells.

Protodioscin isolated from fenugreek (Trigonella foenumgraecum L.) induces cell death and morphological change indicative of apoptosis in leukemic cell line H-60, but not in gastric cancer cell line KATO III.

Protodioscin (PD) was purified from fenugreek (Trigonella foenumgraecum L.) and identified by Mass, and 1H- and 13C-NMR. The effects of PD on cell viability in human leukemia HL-60 and human stomach cancer KATO III cells were investigated. PD displayed strong growth inhibitory effect against HL-60 cells, but weak growth inhibitory effect on KATO III cells. Morphological change showing apoptotic bodies was observed in the HL-60 cells treated with PD, but not in KATO III cells treated with PD. Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 75.2, 96.3, and 100% after a 3-day treatment with 2.5, 5, and 10 microM PD, respectively. The fragmentation by PD of DNA to oligonucleosomal-sized fragments, that is a characteristic of apoptosis, was observed to be both concentration- and time-dependent in the HL-60 cells. These findings suggest that growth inhibition by PD of HL-60 cells results from the induction of apoptosis by this compound in HL-60 cells.