Effects of short-term fructooligosaccharide intake on equol production in Japanese postmenopausal women consuming soy isoflavone supplements: a pilot study.

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 (5 g/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.

Lactoferrin conjugated with 40-kDa branched poly(ethylene glycol) has an improved circulating half-life.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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)].

Trypanocidal constituents in plants: 7. Mammea-type coumarins.

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.

TLR7 mediated viral recognition results in focal type I interferon secretion by dendritic cells.

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.

Antibacterial activity of crude extracts from Mexican medicinal plants and purified coumarins and xanthones.

Thirty-two extracts from 22 Mexican medicinal plants of 15 different families were assayed to determine their antibacterial activity against Escherichia coli and Staphylococcus aureus. Seventeen plants showed antibacterial activity, while five plants showed no activity against both bacteria. All of the extracts showed higher activity against Staphylococcus aureus (methicillin-sensitive and methicillin-resistant) than against Escherichia coli, except one. Among the plants examined, Bursera simaruba (L.) Sarg. (Burseraceae), Haematoxylum brasiletto H. Karst. (Fabaceae), Calophyllum brasiliense Cambess. (Clusiaceae), and Mammea americana L. (Clusiaceae) were highly active against Staphylococcus aureus. Coumarins (mammea A/BA and mammea A/AA) and xanthones, namely jacareubin and 1,3,5,6-tetrahydroxy-2-(3,3-dimethylallyl) xanthone, were isolated as the principle compounds from the last two plants.

Arl8/ARL-8 functions in apoptotic cell removal by mediating phagolysosome formation 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.

Resistant starch promotes equol production and inhibits tibial bone loss in ovariectomized mice treated with daidzein.

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 ß-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.

Trypanocidal constituents in plants 6. 1) Minor withanolides from the aerial parts of Physalis angulata.

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)-15alpha-acetoxy-5alpha,6beta,14beta,17beta,27-pentahydroxy-1-oxo-witha-2, 24-dienolide, (20S,22S)-15alpha-acetoxy-5alpha,6beta,14alpha,23beta-tetrahydroxy-1-oxo-witha-2,16,24-trienolide and (20S,22R)-15alpha-acetoxy-5beta,6beta-epoxy-14alpha-hydoxy-3beta-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.

Bisabolane-type sesquiterpenes from the aerial parts of Lippia dulcis.

Six new bisabolane-type sesquiterpenes, peroxylippidulcines A-C (3-5), peroxyepilippidulcine B (6), and epilippidulcines B (7) and C (8), have been isolated from the aerial parts of Lippia dulcis, along with two known bisabolane-type sesquiterpenes, seven known flavonoids, and a known triterpenoid. The structures of 3-8 were characterized on the basis of NMR, MS, specific rotation, and X-ray crystallographic analysis data and chemical evidence.

Trypanocidal constituents in plants 5. Evaluation of some Mexican plants for their trypanocidal activity and active constituents in the seeds of Persea americana.

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 71 kinds of methanolic and other organic extracts from 65 plants, which were newly examined by a preliminary screening test to observe immobilization of epimastigotes and trypomastigotes of T. cruzi in vitro. The MeOH extract of seeds of Persea americana (avocado) showed moderate activity against epimastigotes. In order to identify the principal compounds for the activity, the MeOH extract was subjected to bioassay-guided fractionation. From the active fractions, six 1,2,4-trihydroxyheptadecane derivatives and two 1,2,4-trihydroxynonadecane derivatives including a new one were isolated. These compounds showed moderate activity against epimastigotes and trypomastigotes.

Antiproliferative constituents in plants 9. Aerial parts of Lippia dulcis and Lippia canescens.

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).

Trypanocidal constituents in plants 4. Withanolides from the aerial parts of Physalis angulata.

The constituents of the aerial parts of Physalis angulata (Solanaceae) were investigated based on the plant's trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent for Chagas' disease. Four new withanolides were isolated, along with six known ones, from the active fraction. Their structures were determined by spectroscopic analysis. Trypanocidal activity against trypomastigotes, an infectious form of T. cruzi, was also estimated, as well as cytotoxic activity against human uterine carcinoma (HeLa) cells in vitro. Evaluation of trypanocidal activity using the colorimetric reagent Cell Counting Kit-8 was also examined.

Antiproliferative constituents in plants 10. Flavones from the leaves of Lantana montevidensis Briq. and consideration of structure-activity relationship.

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.

Ursolic acid as a trypanocidal constituent in rosemary.

The MeOH extract of the leaves of rosemary (Rosmarinus officinalis L.) completely inhibited the motility of cultured epimastigotes of Trypanosoma cruzi at the concentration of 2 mg/ml after 2 h of incubation. Activity-guided fractionation of the MeOH extract has resulted in the isolation of three triterpene acids, betulinic, oleanolic and ursolic acids. Ursolic acid stopped the movement of all T. cruzi epimastigotes at the minimum concentration (MC(100)) of 40 micro g/ml (88 micro M) after 48 h of incubation. Oleanolic acid was less active (MC(100): 250 micro g/ml, 550 micro M) and betulinic acid was practically inactive.

Trypanocidal constituents in plants 2.xanthones from the stem bark of Garcinia subelliptica.

The constituents of the stem bark of Garcinia subelliptica (Guttiferae) were investigated based on its trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent for Chagas' disease. As the active components, nine xanthones were isolated including two new ones, 4-hydroxybrasilixanthone B and 1,3,5,6-tetrahydroxy-4,7,8-tri(3-methyl-2-butenyl)xanthone. Their structures were determined by spectroscopic analysis. Trypanocidal activity against trypomastigotes, an infectious form of T. cruzi, was also estimated as well as cytotoxic activity. Fukugetin, the major component of the bark, showed no activity.

Conduritols as oviposition stimulants for the danaid butterfly, Parantica sita, identified from a host plant, Marsdenia tomentosa.

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.

In vitro cytotoxic activity of phenanthroindolizidine alkaloids from Cynanchum vincetoxicum and Tylophora tanakae against drug-sensitive and multidrug-resistant cancer cells.

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.

Trypanocidal constituents in plants 3. Leaves of Garcinia intermedia and heartwood of Calophyllum brasiliense.

The constituents of the leaves of Garcinia intermedia and heartwood of Calophyllum brasiliense were investigated based on their trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent of Chagas' disease. As the active components, the polyisoprenylated benzophenone derivative guttiferone A and the xanthone 8-desoxygartanin were isolated along with the biflavonoids podocarpusflavone A and amentoflavone, and friedelin from the former. Three xanthones, jacareubin, 6-deoxyjacareubin, and 1,3,5,6-tetrahydroxy-2-(3-methyl-2-butenyl)xanthone from the latter showed activity. The trypanocidal activity of these compounds against trypomastigotes, an infectious form of T. cruzi, was examined as well as gossypol, berberine chloride, and harmine for comparison.

HIV-1 inhibitory compounds from Calophyllum brasiliense leaves.

The hexane, acetone and methanol extracts of Calophyllum brasiliense leaves were fractionated following a three bioassay guide: high HIV-1 RT inhibition, low cytotoxicity on MT2 cells and high inhibition of HIV-1 IIIb/LAV replication. This led to the isolation of three anti HIV-1 dipyranocoumarins: calanolides A and B and soulattrolide. In contrast, other isolated compounds such as apetalic acid, isoapetalic acid, a structural isomer of isoapetalic acid, friedelin, canophyllol and amentoflavone were devoid of HIV-1 RT inhibitory activity. Calanolide C was also obtained as a natural product and showed moderate inhibitory properties.