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Plasmacytoid dendritic cells (pDC) sense viral RNA through toll-like receptor 7 (TLR7), form self-adhesive pDC–pDC clusters, and produce type I interferons. This cell adhesion enhances type I interferon production, but little is known about the underlying mechanisms. Here we show that MyD88-dependent TLR7 signaling activates CD11a/CD18 integrin to induce microtubule elongation. TLR7+ lysosomes then become linked with these microtubules through the GTPase Arl8b and its effector SKIP/Plekhm2, resulting in perinuclear to peripheral relocalization of TLR7. The type I interferon signaling molecules TRAF3, IKKα, and mTORC1 are constitutively associated in pDCs. TLR7 localizes to mTORC1 and induces association of TRAF3 with the upstream molecule TRAF6. Finally, type I interferons are secreted in the vicinity of cell–cell contacts between clustered pDCs. These results suggest that TLR7 needs to move to the cell periphery to induce robust type I interferon responses in pDCs., Antiviral immune responses involve clustering of plasmacytoid dendritic cells (pDC) in response to endosomal TLR7-mediated sensing of viral RNA. Here the authors show the GTPase Arl8b controls translocation of TLR7+ endosomes to the periphery of the cell via microtubule interactions, thus enabling pDC clustering and type I interferon production.

The engulfment and clearance of apoptotic cells by neighboring cells or professional phagocytes is crucial to tissue homeostasis and the regulation of immune responses. The Arf-like GTPase Arl8, which localizes primarily to lysosomes, mediates phagolysosome formation to promote the efficient degradation of apoptotic germ cells in Caenorhabditis elegans., Efficient clearance of apoptotic cells by phagocytes is important for development, tissue homeostasis, and the prevention of autoimmune responses. Phagosomes containing apoptotic cells undergo acidification and mature from Rab5-positive early to Rab7-positive late stages. Phagosomes finally fuse with lysosomes to form phagolysosomes, which degrade apoptotic cells; however, the molecular mechanism underlying phagosome–lysosome fusion is not fully understood. Here we show that the Caenorhabditis elegans Arf-like small GTPase Arl8 (ARL-8) is involved in phagolysosome formation and is required for the efficient removal of apoptotic cells. Loss of function of arl-8 results in the accumulation of apoptotic germ cells. Both the engulfment of the apoptotic cells by surrounding somatic sheath cells and the phagosomal maturation from RAB-5- to RAB-7-positive stages occur in arl-8 mutants. However, the phagosomes fail to fuse with lysosomes in the arl-8 mutants, leading to the accumulation of RAB-7-positive phagosomes and the delayed degradation of apoptotic cells. ARL-8 localizes primarily to lysosomes and physically interacts with the homotypic fusion and protein sorting complex component VPS-41. Collectively our findings reveal that ARL-8 facilitates apoptotic cell removal in vivo by mediating phagosome–lysosome fusion during phagocytosis.

Calophyllum brasiliense and Mammea americana (Clusiaceae) are two trees from the tropical rain forests of the American continent. A previous screening showed high trypanocidal activity in the extracts of these species. Several mammea-type coumarins, triterpenoids and biflavonoids were isolated from the leaves of C. brasiliense. Mammea A/AA was obtained from the fruit peels of M. americana. These compounds were tested in vitro against epimastigotes and trypomastigotes of Trypanosoma cruzi, the etiologic agent of Chagas disease. The most potent compounds were mammea A/BA, A/BB, A/AA, A/BD and B/BA, with MC100 values in the range of 15 to 90 microg/ml. Coumarins with a cyclized gamma,gamma-dimethylallyl substituent on C-6, such as mammea B/BA, cyclo F + B/BB cyclo F, and isomammeigin, showed MC100 values > 200 microg/ml. Several active coumarins were also tested against normal human lymphocytes in vitro, which showed that mammea A/AA and A/BA were not toxic. Other compounds from C. brasiliense, such as the triterpenoids, friedelin, canophyllol, the biflavonoid amentoflavone, and protocatechuic and shikimic acids, were inactive against the epimastigotes. The isopropylidenedioxy derivative of shikimic acid was inactive, and its structure was confirmed by X-ray diffraction. Our results suggest that mammea-type coumarins could be a valuable source of trypanocidal compounds.

We measured the characteristics of trees and sites of 14 Northern Goshawk (Accipiter gentilis) and 14 Eurasian Sparrowhawk (Accipiter nisus) nests in the Ishikari Plain of Hokkaido, Japan, where their nesting habitats were fragmented into small woodlots. We also randomly located 25 plots (0.033 ha each) in unused woodlots to compare differences between nest and random sites. Goshawks nested in a variety of trees and diameters at breast height (dbh) of nest trees were significantly larger than the mean values for all trees in the nest sites, and were similar to the largest trees in these sites. The heights of nest trees were significantly larger than the mean values of all trees in the nest sites. Goshawks nested in a variety of forest types, in proportion to their availability. The dbh and tree heights of sparrowhawk nest trees were significantly larger than the mean values of all trees in the nest sites, and were significantly smaller than the largest trees in these sites. Sparrowhawks nested on...

Host-plant chemicals responsible for egg-laying by the chestnut tiger butterfly, Parantica sita, were identified from one of its hosts, Marsdenia tomentosa. Ovipositing females responded positively to a methanolic extract of the plant. Solvent partitioning of the extract and oviposition bioassays indicated that the active principle resided in the aqueous fraction. Further activity-directed fractionation of the water-soluble constituents by various forms of column chromatography led to the isolation of several saturated and unsaturated cyclitols together with their glycosides. Of these, conduritol A, a predominant cyclitol present in the plant, moderately stimulated oviposition, while conduritol F 2-O-glucoside, although present in a very small amount, evoked a stronger response from females than conduritol A when tested at the same dose. In contrast, its aglycone, conduritol F, which was also a trace component, was almost inactive by itself. However, the oviposition-stimulatory activity of conduritol A was significantly enhanced when tested in combination with a small quantity of conduritol F. Addition of a small quantity of conduritol F 2-O-glucoside to conduritol A resulted in a substantial elevation in female responses. Consequently, the synergistic action of a large amount of conduritol A and small amounts of co-occurring conduritol F and its glucoside can account for the stimulation of egg-laying by P. sita on M. tomentosa.

Two known phenanthroindolizidine alkaloids, (-)-(R)-13aalpha-antofine (1) and (-)-(R)-13aalpha-6-O-desmethylantofine (2), and two new natural products, (-)-(R)-13aalpha-secoantofine (3) and (-)-(R)-13aalpha-6-O-desmethylsecoantofine (4), were isolated from Cynanchum vincetoxicum. The structures of all compounds were established by means of NMR methods including COSY, NOESY, HSQC, and HMBC experiments, supported by HRMS and optical rotation data. Cytotoxic activity of the isolated alkaloids, and of three other alkaloids previously isolated from Tylophora tanakae, (-)-(R)-13aalpha-tylophorine (5), (-)-(R)-13aalpha-7-O-desmethyltylophorine (6), and (+)-(S)-13abeta-isotylocrebrine (7), was assessed in vitro using a drug-sensitive KB-3-1 and a multidrug-resistant KB-V1 cancer cell line. Structure-activity relationships in this series of alkaloids are discussed. The IC(50) values of some of the alkaloids are in the low nanomolar range, being thus comparable to the activity of clinically used cytotoxic drugs. Previously reported adverse side effects of these alkaloids could possibly be overcome by modern tissue-specific drug targeting techniques.

The flavonoid fraction from the leaves of Lantana montevidensis Briq. (Verbenaceae) showed antiproliferative activity against human gastric adenocarcinoma (MK-1, GI50: 12 microg/ml), human uterus carcinoma (HeLa, 5 microg/ml), and murine melanoma (B16F10, 5 microg/ml) cells in vitro. Bioactivity-guided chemical investigation of the fraction has resulted in the isolation of apigenin (10) and ten 5,6,7-oxygenated flavones: cirsilineol (1), eupatorin (2), 5,4'-dihydroxy-6,7,3',5'-tetramethoxyflavone (3), 5,6-dihydroxy-7,3',4'-trimethoxyflavone (4), 5,6,4'-trihydroxy-7,3',5'-trimethoxyflavone (5), 5,6,3'-trihydroxy-7,4'-dimethoxyflavone (6), 5,3',4'-trihydroxy-6,7,5'-trimethoxyflavone (7), cirsiliol (8), hispidulin (9), and eupafolin (11). Antiproliferative activity of the isolated flavones, some other related flavones (luteolin, baicalein, 6-hydroxyluteolin, pectolinarigenin, jaceosidin, desmethoxycentaureidin, eupatilin, and chrysin) from other plant materials, and synthetic 6- and 7-methoxyflavones was evaluated, and the structure-activity relationships were examined.

Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosoma cruzi, which is the etiological agent for Chagas' disease, one of the most serious protozoan diseases in Latin America. There were 43 kinds of methanolic and other organic extracts from 39 plants which were examined by the preliminary screening test to see immobilization of epimastigotes of T. cruzi in vitro. Eighteen of them showed activity at the concentration of 2 mg/ml after incubation for 2 h, while 13 showed activity at the concentration of 1 mg/ml after incubation for 48 h. Among them, the MeOH extract of roots of Aristolochia taliscana (Aristolochiaceae), locally known as "Guaco," immobilized all the epimastigotes even at lower concentration of 0.5 mg/ml (48 h). In order to identify principal compounds for this activity, the MeOH extract of Guaco was subjected to bioassay-guided fractionation. From the active fractions, four neolignans, eupomatenoid-7 (1), licarin A (2), eupomatenoid-1 (5) and licarin B (6), and two lignans, austrobailignan-7 (3) and fragransin E1 (4) were isolated. Compounds 1-4 immobilized all the epimastigotes at the minimum concentration of 25-75 microg/ml after incubation for 48 h, while compounds 5 and 6 were inactive. Corresponding concentration of gossypol, berberine chloride and harmine was 280 microg/ml, 300 microg/ml and >500 microg/ml, respectively.

Summary Oviposition response of Ideopsis similis (L.) (Lepidoptera: Danaidae) was examined for 12 phenanthroindolizidine alkaloids present in its host plant, Tylophora tanakae (Maxim.) (Asclepiadaceae). At least five alkaloids, i.e. (+)-isotylocrebrine (3,4,6,7-tetramethoxyphenanthroindolizidine; l), (+)-3-demethyliso- tylocrebrine (3), (+)-isotylocrebrine N-oxide (5), (+)-6-demethyltylocrebrine (8) and (–)-7-demethyltylophorine (10), were found to individually stimulate oviposition by females. Of these, compounds 1, 3 and 10 were regarded as key components most responsible for host recognition or preference. However, female egg-laying was much higher in response to a mixture of the five alkaloids. In two-choice bioassays, more eggs were deposited on samples comprising the five alkaloids than on samples consisting of a single alkaloid. This suggests strongly that host selection by the butterfly is mediated by the synergistic action of several phenanthroindolizidine alkaloids present in the host plant.

Sixteen glycosides of polyoxypregnanes, including two new pregnanes, isokidjoladinin and isotemontosin, were isolated form the leaves and caules of Marsdenia tomentosa collected in Fukuoka. The structures of fourteen new glycosides were elucidated and named marstomentosides A-N.

Five new glycosides of 14, 15-seco- and 13, 14 : 14, 15-diseco-type pregnanes, including a new pregnane, 2α-hydroxyhirundigenin, were isolated, in addition to one known glycoside, cynatratoside B, from the roots of Tylophora tanakae MAXIM. Their structures were elucidated by spectral and chemical means.

Pregnanes in Asclepiadaceae plants have been studied actively since the 1960s, and many pregnanes and pregnane glycosides were disclosed in several genera. Hoya carnosa R. BR. is one of the liana belonging to Asclepiadaceae and grown in the Ryukyu district. The pregnane ingredients in this plant, however, have not yet been investigated, although cyclitols were studied. 1) As a part of our investigation of the steroidal constituents from Apocynaceae 2) and Asclepiadaceae 3) plants, pregnanes and pregnane glycosides from the caules of this plant were examined in advance of those from the leaves. When the air-dried caules of H. carnosa were percolated with MeOH and the MeOH extract was partitioned with CHCl3‐H2O, pregnan glycosides were observed in the CHCl3 fraction. After a portion of the CHCl 3 extract was hydrolyzed under mild conditions, pregnanes and sugars were isolated using a silica gel column. From the pregnane fraction, eleven pregnanes (a-1—a-11) were obtained. Five known pregnanes were identified to be drevogenin P (a-1), 4) drevogenin A (a-3), 4) 17b-marsdenin (a-4), 5) drebyssogenin J (a-8) 6) and marsectohexol (a-9), 7) based on comparisons of the NMR and MS data with those in the literature. The high resolution (HR)-FAB-MS of a-2 suggested the molecular formula C25H36O7, which was two acetyl residues greater than a-1. The 13 C-NMR signals showed a similar pattern to those of a-1 and a-3. The presence of acetyl groups was confirmed by two 3H-singlet signals in the 1 H-NMR spectrum (d 1.96, 2.10). In a-2, two carbinyl protons observed at d 3.67 (t, J510 Hz) and d 3.05 (d, J510 Hz) in a-1 were shifted to d 5.36 (t, J510 Hz) and d 4.83 (d, J510 Hz), respectively, with almost the same chemical shifts as those in 11,12-diacylated pregnanes such as a-3. The structure of a-2 was thus assigned to be 11,12-di-O-acetyldrevogenin P. The relation similar to that between a-2 and a-1 was seen in comparison of the 1 H-NMR spectra between a-5 and a-4. The presence of two acetyl groups was suggested in a-5, based on two 3H-singlet signals at d 1.99 and 2.09, and also on the lower field shifts of two carbinyl protons in comparison with those of a-4 (Dd51.62, 1.74 ppm, respectively). During the NMR measurement, the H-21 signal became am

Four cyclitol glycosides and a new tetrol and pentol, were obtained, along with five known cyclitols from the fresh leaves of Marsdenia tomentosa . The locations of the glycosidic linkage in the cyclitols were determined by spectral and chemical methods.

Seventeen alkaloids were isolated from the air-dried leaves of Alstonia villosa and their structures characterized. One of the seven new alkaloids was elucidated to be (19Z)-5α-methoxyrhazimine, having a 3, 4-dihydroquinoline nucleus.

Further study of the methanol extract of the aerial parts of Physalis angulata (Solanaceae) resulted in the isolation of new withanolides, designated physagulins L, M and N, together with known withanolide, physagulin D and flavonol glycoside, quercetin 3-O-rhamnosyl-(1→6)-galactoside. The chemical structures of these new withanolides were elucidated by detailed spectroscopic analyses to be (20R,22R)-15α-acetoxy-5α,6β,14β,17β,27-pentahydroxy-1-oxo-witha-2, 24-dienolide, (20S,22S)-15α-acetoxy-5α,6β,14α,23β-tetrahydroxy-1-oxo-witha-2,16,24-trienolide and (20S,22R)-15α-acetoxy-5β,6β-epoxy-14α-hydoxy-3β-methoxy-1-oxo-witha-16,24-dienolide, respectively. All these compounds showed weak trypanocidal activity against trypomastigotes, an infectious form of Trypanosoma cruzi, the etiologic agent for Chagas' disease. Withanolides obtained in the previous paper showed considerable trypanocidal activity, suggesting the structure–activity relationships.

A giant danaid butterfly, Idea leuconoe, specializes on apocynaceous plants such as Parsonsia laevigata, which has been reported to contain pyrrolizidine alkaloids. Females of I. leuconoe deposited eggs in response to methanolic extract of P. laevigata, and subsequent bioassay-guided fractionation of the extract revealed that phytochemicals crucial for host recognition by ovipositing females are Parsonsia-specific macrocyclic pyrrolizidine alkaloids including parsonsianine, parsonsianidine, and 17-methylparsonsianidine. Parsonine, another P. laevigata pyrrolizidine component with a keto-dihydropyrrolizine moiety that is closely related in structure to male pheromones of the butterfly, and several nonhost pyrrolizidine alkaloids were entirely inactive. We interpret these data as strong evidence for an ancestral association through herbivory between danaid butterflies and pyrrolizidine alkaloids.

On examination of the less polar fraction from the bark of Wikstroemia retusa, nine daphnane-type diterpenoids, including four known ones, were isolated and the structures established.

Background Recent studies suggest that some of the clinical effectiveness of soy or daidzein, which is a type of isoflavone, may be attributed to a person’s ability to produce equol from daidzein. Equol, which is a metabolite of one of the major soybean isoflavones called daidzein, is produced in the gastrointestinal tract by certain intestinal microbiota where present. Habitual dietary patterns may alter the intestinal bacterial profile, and influence the metabolism of isoflavones and the production of equol. Fructooligosaccharides (FOS) have a prebiotic activity as well as being a dietary fibre. The purpose of the present study was to determine whether FOS supplementation increases equol production in equol producers and stimulates equol production in equol non-producers in Japanese postmenopausal women. Methods A soy challenge was used to assess equol-producer status prior to the start of the study in healthy postmenopausal Japanese women. The study involved 4 separate groups in randomised crossover design. First, subjects were classified as equol producers (n = 25) or non-producers (n = 18), and then they were randomly assigned to the FOS or control group. All subjects received a daily dose of 37 mg isoflavone conjugates in the capsule (21 mg aglycone form) and either FOS (5g/day) or sucrose as control, in a randomised crossover study design. Equol -production was assessed by testing the serum and urine before and after the 2-week supplementation period. Results The analyses were conducted on 34 subjects completed the study, 21 (61.8%) were classified as equol producers, and 13 (38.2%) as non-producers. Significant differences were observed in the interaction effect of time × equol state after 1 week of intervention (p = 0.006). However there were no effects after 2 weeks of intervention (p = 0.516). Finally, in both equol producers and non-producers, FOS supplementation did not affect the serum equol concentration or the urinary equol to daidzein concentration ratios. Conclusions We have reported that FOS intervention (5 g/day for 2 weeks) does not significantly modulate the capacity of intestinal microbiota to produce equol in postmenopausal Japanese women, in either equol producers or non-producers in this pilot study. Further larger investigations that explore the roles of specific intestinal microbiota in equol production will enable the establishment of dietary conditions that are required to enhance equol production.

10-Carboxyloganin was isolated along with β-D-glucosides of cyclopentano-normonoterpenoids and dinormonoterpenoids from the frozen fresh leaves of Cerbera manghas. Their structures were characterized by means of MS and NMR spectroscopy. The correlation of drying conditions of the leaves and yields of glucosides or allosides of cyclocerberidol and epoxycerberidol was studied. The dinormonoterpenoids were similar to those obtained from Thevetia peruviana.

In crude plant extracts, constituents of biological or pharmaceutical interest often exist in the form of glycosides. Off-line mass spectral investigations of these metabolites require soft ionisation techniques such as desorption chemical ionisation (DCI) or fast atom bombardment (FAB) if information on molecular mass or sugar sequence is desired. In LC-MS, glycosides can be ionised by using thermospray (TSP), continuous-flow fast atom bombardment (CF-FAB) or other interfaces. These techniques are thus potentially applicable to the on-line analysis of glycosides and can be applied to plant extract analysis. Thermospray (TSP) used with ammonium acetate as buffer provides mass spectra similar to those obtained with DCI-MS using NH3 and is potentially applicable to the on-line analysis of relatively small glycosides bearing no more than three sugar units. CF-FAB provides cleaner MS spectra than static FAB due to the lower concentration of the matrix used and can be applied to more polar compounds such as glycosides with a larger number of sugars. The use of a special setup involving post-column addition of the buffer or the matrix and splitting allows LC-UV, TSP LC-MS and CF-FAB LC-MS to be performed with the same standard HPLC conditions. Different crude plant extracts containing various types of glycosides with one to eight sugar units have been analysed by both TSP and CF-FAB. Cardenolides from Nerium odorum (Apocynaceae) and saponins from Swarzia madagascariensis (Leguminosae), Aster scaber and Aster tataricus (Asteraceae) have been studied by LC-MS. The combination of these two interfaces for the HPLC screening of crude plant extracts is discussed.

Chemicals releasing oviposition by an Asclepiadaceae feeder,Ideopsis similis, were identified from a host plant,Tylophora tanakae. A strong positive response was evoked by a methanolic extract of the plant, which proved to contain multiple stimulants. A mixture of two phenanthroindolizidine alkaloids, (+)-isotylocrebrine and (−)-7-demethyltylophorine, isolated from organic fractions, elicited significant ovipositional responses from females.

From the fresh leaves of Tylophora tanakae , ten phenanthroindolizine alkaloids were isolated. Among them, eight were new alkaloids and their structures were determined. Two known alkaloids were identified as isotylocrebrine and tylophorine.

C-nor-D-homo-homologues of cannogenin and uzarigenin glycosides were isolated along with known cardenolide glycosides from the frozen fresh leaves of Thevetia neriifolia. A bisdesmosidic tetraoside of 3 beta,14,21-trihydroxy-5 beta,14 beta-pregnan-20-one was also obtained from the polar fraction and the structure established.

Steroidal constituents from the roots and stems of Asclepias fruticosa L. were investigated separately. From the roots, twelve pregnane pentaosides and uzarigenin beta-sophoroside were isolated together with three known coroglaucigenin and corotoxigenin glycosides. Pregnane glycosides were composed of ikemagenin or kidjolanin as an aglycone, and D-digitoxose, D-cymarose, D-oleandrose and terminal D-glucose as component sugars. Among the constituents from the stems, cardenolides show a similar pattern to those from leaves. 17 alpha-Hydroxycalactin and 17 alpha-hydroxyafroside were newly obtained along with known doubly linked and normally linked glycosides. Two pregnane glycosides and uzarigenin beta-sophoroside obtained from the roots were also isolated.

Steroidal constituents from the roots of Apocynum cannabinum L. were investigated. (20S)-, (20R)-18, 20-Epoxycymarin and (20S)-18, 20-epoxyapocannoside were isolated along with cannogenin, strophanthidin and cannogenol glycosides, including D-cymaroside, D-oleandroside, D-digitoxoside and D-digitaloside, and their glucosyl, cellobiosyl or gentiobiosyl glycosides. Two pregnanes, neridienone A and 6, 7-didehydrocortexone, were obtained, accompanied with cardenolides.

The antiproliferative constituents in the MeOH extracts of the aerial parts of Lippia dulcis Trev. and Lippia canescens Kunth (Verbenaceae) were investigated. Activity-guided chemical investigation of the MeOH extracts resulted in the isolation of the three bisabolane-type sesquiterpenes [(+)-hernandulcin (1), (-)-epihernandulcin (2), and (+)-anymol (3)] and four phenylethanoid glycosides [acteoside (4), isoacteoside (5), martynoside (6), and a new diacetylmartynoside (7)] from the former, and four phenylethanoid glycosides [acteoside (4), isoacteoside (5), arenarioside (8), and leucosceptoside A (9)] and three flavones [desmethoxycentaureidin (10), eupafolin (11), and 6-hydroxyluteolin (12)] from the latter. Antiproliferative activity of the isolated compounds against murine melanoma (B16F10), human gastric adenocarcinoma (MK-1), and human uterine carcinoma (HeLa) cells was estimated. (+)-Anymol (3), acteoside (4), isoacteoside (5), arenarioside (8), eupafolin (11), and 6-hydroxyluteolin (12) had GI50 values of 10-16 microM against B16F10 cell. Desmethoxycentaureidin (10) and eupafolin (11) showed high inhibitory activity against HeLa cell growth (GI50 9 microM, and 6 microM, respectively).

Along with five known alkaloids, N(4)-oxides of cathafoline and 11-methoxyakuammicine, vincamajine 17-O-veratrate and vincamajine N(1)-tri-O-methylgallate were isolated from the leaves of Alstonia macrophylla growing in Thailand. Their structures were determined by spectral methods.

The leaves of Alstonia macrophylla collected in Los Banos, The Philippines, were investigated and five new alkaloids were obtained along with four known alkaloids. The structures were determined by means of mass and NMR spectroscopy.

Daidzein is metabolized to equol in the gastrointestinal tract by gut microflora. Equol has greater estrogenic activity than genistein and daidzein, with its production shown to be promoted by dietary fiber. It is known that resistant starch (RS) is not absorbed in the proximal intestine and acts as dietary fiber in the colon. In this study, we investigated the combined effects of daidzein and RS intake on equol production, bone mineral density, and intestinal microflora in ovariectomized (OVX) mice. Female mice of the ddY strain, aged 8 weeks, were either sham operated (n = 6) or OVX. The OVX mice were randomly divided into 5 groups: OVX control (n = 6), OVX fed 0.1% daidzein-supplemented diet (OVX + Dz, n = 8), OVX fed 0.1% daidzein- and 12% RS-supplemented diet (OVX + Dz + RS, n = 8), OVX fed 12% RS-supplemented diet (OVX + RS, n = 8), and OVX who received daily subcutaneous administration of 17 β-estradiol (n = 6). After 6 weeks, urinary equol concentration was significantly higher in the OVX + Dz + RS group than in the OVX + Dz group. The bone mineral density of the whole tibia was higher in the OVX + Dz +RS group compared with the OVX + Dz group. The occupation ratios of Bifidobacterium spp in the cecal microflora in groups fed RS were significantly higher than those in the other groups. The present study demonstrated that RS may increase the bioavailability of daidzein.

In addition to ten alkaloids previously described, we have isolated two alkaloid N-oxides from Tylophora tanakae MAXIM. (Asclepiadaceae). Further, the polar fraction of the leaves and caules of this plant cultivated in a farm afforded two new polar alkaloids. The structures of the four products were determined. The relation between the structure and cytotoxic activity of this group of alkaloids is discussed.

Seventeen alkaloids were isolated from the air-dried leaves of Alstonia villosa and their structures characterized. One of the seven new alkaloids was elucidated to be (19Z)-5α-methoxyrhazimine, having a 3, 4-dihydroquinoline nucleus.

Sixteen glycosides of polyoxypregnanes, including two new pregnanes, isokidjoladinin and isotemontosin, were isolated form the leaves and caules of Marsdenia tomentosa collected in Fukuoka. The structures of fourteen new glycosides were elucidated and named marstomentosides A-N.

Cardenolide glycosides from the seeds of Anodendron affine were reinvestigated. Five doubly linked cardenolide glycosides, designated affinosides P-T, and three normally linked sarmentogenin and affinogenin A glycosides were newly isolated, along with affinosides A, K, M and other known glycosides.

Following the investigation of the constituents from the leaves of Thevetia neriifolia cultivated in Kumamoto, Japan, the leaves of the same species collected in Singapore were examined, and C-nor- d -homocardenolide glycosides were obtained as in the preceding study, along with other common glycosides. α- l -Rhamnosides of digitoxigenin, cannogenin and thevetiogenin, and glycosides of uzarigenin were isolated from the leaves. Two 18,20-epxoy-derivatives of digitoxigenin α- l -thevetoside were isolated and the C-20 configurations are discussed.

Polar glycosides from the air-dried leaves were re-examined, and gentiobiosyl-nerigoside and gentiobiosylbeaumontoside isolated along with the major trioside, gentiobiosyl-oleandrin. Minor triosides also include glycosides of8β-hydroxy- and Δ 16 -8β-hydroxy-digitoxigenin, and Δ 16 -neriagenin, along with glycosides of known cardenolides, oleandrigenin, digitoxigenin, adynerigenin, neriagenin and their Δ 16 -derivatives.

Along with known digitoxigenin α- l -thevetoside and α- l -acofrioside, the corresponding glycosides of a C -nor- d -homocardenolide were isolated from the air-dried leaves of Thevetia neriifolia and their structures determined by spectral and chemical methods. Triosides of the C -nor- d -homo-cardenolide were obtained from the methanol extract of the fresh leaves.

Conduritol F 3-O- and 4-O-glucosides were obtained from Cynanchum liukiuense, along with conduritol F which was identified in all Asclepiadaceous plants examined, Tylophora tanakae, Asclepias curassavica and A. fruticosa, as well as in Marsdenia tomentosa. The pattern of the glucosidic linkage to conduritol F differed between individual species, 2-O-glucoside from T. tanakae and M. tomentosa, 3-O-glucoside from A. curassavica, but none from A. fruticosa. Along with conduritol F glucosides, an 11-glucosyloxy-megastigmane and a monoterpenoid glucoside were isolated from C. liukiuense.

Steroidal compounds in the roots of Asclepias tuberosa were investigated and 17alpha-hydroxyandrosta-4,6,15-trien-3-one 17-O-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside, termed ascandroside, was isolated from the CHCl3-soluble fraction. Among five doubly-linked cardenolide glycosides, two were identified as 3'-spiro-linked thiazolidinone (4) and S-oxythiazolidinone derivatives (5) of delta5-calotropin. The stereochemistry at the C-3' in these two cardenolides is discussed. 3'-O-beta-D-Glucopyranosyl-delta5-calotropin (3) was also isolated along with A5-calotropin and its 3'-acetate. Nine glycosides of uzarigenin, coroglaucigenin and corotoxigenin were identified.

We developed a lactoferrin conjugate by modifying bovine lactoferrin (bLF) with a 40-kDa branched poly(ethylene glycol) (PEG) molecule (designated 40 k-PEG-bLf), and we evaluated its in vitro activities and pharmacokinetic properties.We prepared 40k-PEG-bLf by amino conjugation with N-hydroxysuccinimide-activated PEG. This conjugate was purified by cation exchange chromatography and its in vitro biological activities, such as iron binding, anti-inflammatory effects, and resistance to proteolytic enzymes were investigated. In vivo pharmacokinetics analyses, were also performed to examine the rate of clearance from the plasma in rats.The 40k-PEG-bLf conjugate was fully active in iron binding and exhibited 97.1 +/- 5.5% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The in vitro peptic susceptibility of 40 k-PEG-bLf revealed that the proteolytic half-life increased at least 6-fold that of unmodified LF. This PEGylated conjugate demonstrated a plasma half-life that was 8.7-fold longer than that of the unmodified bLF in rats.The 40k-PEG-bLf exhibited improved in vitro bioactivity and stability and enhanced pharmacokinetic properties as compared to those of the unmodified bLF and the 20 k-PEG-bLf conjugate, which was recently developed by PEGylation of bLF with a 20-kDa branched PEG [Nojima Y. et al. Bioconjugate Chem. 19:2253-2259 (2008)].

The LEC strain of rats that spontaneously develops hepatic injury has been introduced into specific pathogen-free (SPF) conditions (SPF-LEC/Otk). The present communication describes the clinical and pathological features of the SPF-LEC/Otk rats. The characteristic features of these animals are as follows: (i) Jaundice develops in almost all rats with increase in the P-GPT level; (ii) The animals show episodes of jaundice, a high P-GPT level and liver cell necrosis, but only slight inflammatory cell infiltration; (iii) The liver cells show characteristic microvesicular fatty changes; (iv) The P-GPT level shows increases, first at 18 weeks and then at 25 weeks of age; (v) The rats show immunological disorders, such as deficiency of immunoglobulins, especially IgG1, and of helper T cells; (vi) Infectious agents such as viruses do not seem to be involved, although this possibility cannot be absolutely excluded; (vii) The immunological disorders are not directly associated with the occurrence of liver cell necrosis; and (viii) The pattern of inheritance (autosomal single-recessive trait) of the disease strongly suggests that it is due to a genetic metabolic disorder.

Three new iridals were isolated, along with two known compounds; (+)-6R,10S,11S,14S,26R)-26-hydroxy-15-methylidenespiroirid-16-enal and iso-iridogermanal, from the rhizomes and roots of Belamcanda chinensis and Iris japonica. The new iridals were determined to be 28-acetoxy-14,15-dihydro-26-hydroxy-19-methylidenespiroirida-15,17-dienal (belamcandal), its deacetyl derivative and 16-O-acetyl iso-iridogermanal. Their fatty acid esters were also isolated. Belamcandal stimulates throat membrane.

The thermal degradation of cardenolide glycosides consisting of different sugar moieties and aglycones was examined. On simple heating, the sugar—aglycone linkage of cardenolide glycosides having 2,6-dideoxy sugar moieties in readily cleaved, to afford their genuine aglycones. On the contrary, cardenolide glycosides possessing 6-deoxy sugar moieties are resistant to the degradative reaction. Besides the fission of the glycosidic linkages, some interesting reactions also took place. The pyrolyzed products were isolated by chromatography, and the structures were elucidated by spectroscopic evidence.

Lagunamine (19-hydroxytubotaiwine), angustilobine B acid and losbanine (6,7- seco -6- nor -angustilobine B) were obtained from the leaves of Philippine Alstonia scholaris , together with tubotaiwine, its oxide, and 6,7- seco - angustilobine B; the structures were determined from spectral evidence. 17- O -Acetylechitamine was isolated from the bark of the plant along with echitamine. Alkaloidal patterns in the leaves and barks of Philippine A. scholaris are discussed in relation to those reported from the same plant growing in Asian continental districts.