Development of LC-MS/MS analysis of cyclic dipeptides and its application to tea extract.

2,5-Diketopiperazines (DKPs), also called cyclic dipeptides, have been known to occur in various foods. Recently, DKPs have attracted attentions as bioactive components. There were some reports on analytical methods for DKPs, but the number of analyzed DKPs was only a part of all DKPs and the quantitative performance was not studied in detail. In this study, we selected 31 kinds of DKPs and developed a quantitative and simultaneous analytical method using LC-MS/MS. This method was applied to DKPs determination in Pu-erh tea, post-fermentation tea, and 18 kinds of DKPs were determined at concentration of 0.0017-0.11 ppm. As a result of spiked test, it was concluded that the developed method using LC-MS/MS was useful for estimating DKPs concentration in tea.

Simultaneous evaluation of one-electron reducing systems and radical reactions in cells by nitroxyl biradical as probe.

In the present study, a novel probe for the simultaneous evaluation of one-electron reducing systems (electron transport chain) and one-electron oxidizing systems (free radical reactions) in cells by electron chemical detection was developed. Six-membered cyclic nitroxyl radicals (2,2,6,6-tetramethylpiperidine-1-oxyl; TEMPO series) are sensitive to one-electron redox systems, generating the hydroxylamine form [TEMPO(H)] via one-electron reduction, and the secondary amine form [TEMPO(N)] via one-electron oxidation in the presence of thiols. In contrast, the sensitivities of five-membered cyclic nitroxyl radicals (2,2,5,5-tetramethylpyrrolidine-1-oxyl; PROXYL series) to the one-electron redox systems are comparatively low. The electron chemical detector can detect 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), TEMPO(H) and PROXYL but not TEMPO(N). Therefore, nitroxyl biradical, TEMPO-PROXYL, as a probe for the evaluation of one-electron redox systems was employed. TEMPO-PROXYL was synthesized by the conjunction of 4-amino-TEMPO with 3-carboxyl-PROXYL via the conventional dicyclohexyl carbodiimide reaction. TEMPO-PROXYL, TEMPO(H)-PROXYL and TEMPO(N)-PROXYL were simultaneously quantified by HPLC with Coularray detection. Calibration curves for the quantification of TEMPO-PROXYL, TEMPO(H)-PROXYL and TEMPO(N)-PROXYL were linear in the range from 80 nm to 80 µm, and the lowest quantification limit of each molecule was estimated to be <80 nm. The relative standard deviations at 0.8 and 80 µm were within 10% (n = 5). Copyright © 2015 John Wiley & Sons, Ltd.

[Sudden death associated with myocardial damage caused by microthrombi in a patient with thrombotic thrombocytopenic purpura].

We describe a 35-year-old woman with Down's syndrome who was admitted to a clinic with anorexia and vomiting. Since laboratory findings showed anemia (Hb 7.4 g/dl) and thrombocytopenia (0.5 × 104/µl), she was transferred to our hospital for treatment. Further laboratory examinations revealed schistocytes, LDH elevation, and a negative Coombs' test. Thrombotic thrombocytopenic purpura (TTP) was suspected. Plasma exchange (PEX) and prednisolone administration were thus immediately initiated. Prior to these treatments, ADAMTS13 activity was less than 5% and inhibitors were detected at a level of 0.8 Bethesda U/ml. Although her platelet count had risen to 13.0 × 104/µl by day 6 (post 4 sessions of PEX), it had decreased to 1.8 × 104/µl on day 7. Despite ongoing PEX, thrombocytopenia persisted. On day 21, she suddenly died. Autopsy findings revealed no evidence of myocardial necrosis or coronary artery thrombosis. Extensive microthrombi were, however, detected in precapillary arterioles, capillaries, and post-capillary venules of the heart. Therefore, this patient's sudden death was clinically suspected to have been caused by cardiomyopathy, which had produced cardiogenic shock.

10-Hydroxy-2-decenoic Acid, the Major Lipid Component of Royal Jelly, Extends the Lifespan of Caenorhabditis elegans through Dietary Restriction and Target of Rapamycin Signaling.

Royal jelly (RJ) produced by honeybees has been reported to possess diverse health-beneficial properties and has been implicated to have a function in longevity across diverse species as well as honeybees. 10-Hydroxy-2-decenoic acid (10-HDA), the major lipid component of RJ produced by honeybees, was previously shown to increase the lifespan of Caenorhabditis elegans. The objective of this study is to elucidate signaling pathways that are involved in the lifespan extension by 10-HDA. 10-HDA further extended the lifespan of the daf-2 mutants, which exhibit long lifespan through reducing insulin-like signaling (ILS), indicating that 10-HDA extended lifespan independently of ILS. On the other hand, 10-HDA did not extend the lifespan of the eat-2 mutants, which show long lifespan through dietary restriction caused by a food-intake defect. This finding indicates that 10-HDA extends lifespan through dietary restriction signaling. We further found that 10-HDA did not extend the lifespan of the long-lived mutants in daf-15, which encodes Raptor, a target of rapamycin (TOR) components, indicating that 10-HDA shared some longevity control mechanisms with TOR signaling. Additionally, 10-HDA was found to confer tolerance against thermal and oxidative stress. 10-HDA increases longevity not through ILS but through dietary restriction and TOR signaling in C. elegans.

Dynamics of different bacterial communities are capable of generating sustainable electricity from microbial fuel cells with organic waste.

The relationship between the bacterial communities in anolyte and anode biofilms and the electrochemical properties of microbial fuel cells (MFCs) was investigated when a complex organic waste-decomposing solution was continuously supplied to MFCs as an electron donor. The current density increased gradually and was maintained at approximately 100 to 150 mA m(-2). Polarization curve analyses revealed that the maximum power density was 7.4 W m(-3) with an internal resistance of 110 Ω. Bacterial community structures in the organic waste-decomposing solution and MFCs differed from each other. Clonal analyses targeting 16S rRNA genes indicated that bacterial communities in the biofilms on MFCs developed to specific communities dominated by novel Geobacter. Multidimensional scaling analyses based on DGGE profiles revealed that bacterial communities in the organic waste-decomposing solution fluctuated and had no dynamic equilibrium. Bacterial communities on the anolyte in MFCs had a dynamic equilibrium with fluctuations, while those of the biofilm converged to the Geobacter-dominated structure. These bacterial community dynamics of MFCs differed from those of control-MFCs under open circuit conditions. These results suggested that bacterial communities in the anolyte and biofilm have a gentle symbiotic system through electron flow, which resulted in the advance of current density from complex organic waste.

Identification of the phenolic compounds contributing to antibacterial activity in ethanol extracts of Brazilian red propolis.

The purpose of this study is to identify the quantity and antibacterial activity of the individual phenolic compounds in Brazilian red propolis. Quantitative analysis of the 12 phenolic compounds in Brazilian red propolis was carried out using reversed-phase high-performance liquid chromatography. The main phenolic compounds in Brazilian red propolis were found to be (3S)-vestitol (1), (3S)-neovestitol (2) and (6aS,11aS)-medicarpin (4) with quantities of 72.9, 66.9 and 30.8 mg g of ethanol extracts(- 1), respectively. Moreover, the antibacterial activities of each compound against Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa were evaluated by measuring the minimum inhibitory concentrations. In particular, compound 4 exhibited the most potent antibacterial activity among all the assayed compounds against selected bacteria, indicating that 4 is the most active compound in Brazilian red propolis extracts. Thus, Brazilian red propolis may be used as food additives and pharmaceuticals to protect against bacteria.

Electricity producing property and bacterial community structure in microbial fuel cell equipped with membrane electrode assembly.

It is important for practical use of microbial fuel cells (MFCs) to not only develop electrodes and proton exchange membranes but also to understand the bacterial community structure related to electricity generation. Four lactate fed MFCs equipped with different membrane electrode assemblies (MEAs) were constructed with paddy field soil as inoculum. The MEAs significantly affected the electricity-generating properties of the MFCs. MEA-I was made with Nafion 117 solution and the other MEAs were made with different configurations of three kinds of polymers. MFC-I equipped with MEA-I exhibited the highest performance with a stable current density of 55 ± 3 mA m⁻². MFC-III equipped with MEA-III with the highest platinum concentration, exhibited the lowest performance with a stable current density of 1.7 ± 0.1 mA m⁻². SEM observation revealed that there were cracks on MEA-III. These results demonstrated that it is significantly important to prevent oxygen-intrusion for improved MFC performance. By comparing the data of DGGE and phylogenetic analyzes, it was suggested that the dominant bacterial communities of MFC-I were constructed with lactate-fermenters and Fe(III)-reducers, which consisted of bacteria affiliated with the genera of Enterobacter, Dechlorosoma, Pelobacter, Desulfovibrio, Propioniferax, Pelosinus, and Firmicutes. A bacterium sharing 100% similarity to one of the DGGE bands was isolated from MFC-I. The 16S rRNA gene sequence of the isolate shared 98% similarity to gram-positive Propioniferax sp. P7 and it was confirmed that the isolate produced electricity in an MFC. These results suggested that these bacteria are valuable for constructing the electron transfer network in MFC.

Laxative effects and mechanism of action of Brazilian green propolis.

BACKGROUND: Brazilian green propolis is reported to have wide range of biological properties including antibacterial, anti-inflammatory, anti-influenza, and antioxidant activities. In the digestive system, a protective effect of propolis on gastric ulcer has been reported, but a laxative effect has not yet been reported. We investigated the effect and the mechanism of action of water and ethanol extracts of Brazilian green propolis. METHODS: We examined the laxative effect of propolis on stool frequency by administering orally an ethanol extract of propolis (EEP) or a water extract of propolis (WEP) at 10, 50, 100, or 500 mg/kg to normal mice. We then investigated the effects of propolis using constipation model mice induced by two types of drugs, loperamide (a µ opioid receptor agonist) and clonidine (an α-2 adrenergic receptor agonist). We also investigated the effects of WEP on gastrointestinal transit and contractional tension of the ileum to uncover the mechanism of action of WEP. RESULTS: Treatment with WEP, but not with EEP, significantly increased the weight of stools (p<0.01 at 500 mg/kg). WEP treatment significantly restored stool frequency and stool weight in clonidine-induced constipation model mice, but not in loperamide-induced constipation model mice. WEP treatment did not affect gastro-intestinal transit, but significantly increased the contractional tension of the isolated ileum of guinea pigs. This increase was inhibited by an acetylcholine receptor antagonist (atropine), but not by a 5-HT receptor antagonist (GR113808). CONCLUSION: These findings indicate that WEP has laxative effects both in normal mice and in clonidine-induced constipation model mice. The laxative effects of WEP might be mediated by increased contractional tension of the ileum exerted at least in part via activation of an acetylcholine receptor.

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

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

Ethanolic extracts of Brazilian red propolis increase ABCA1 expression and promote cholesterol efflux from THP-1 macrophages.

The ATP-binding cassette transporter A1 (ABCA1) is a membrane transporter that directly contributes to high-density lipoprotein (HDL) biogenesis by regulating the cellular efflux of cholesterol. Since ABCA1 plays a pivotal role in cholesterol homeostasis and HDL metabolism, identification of a novel substance that is capable of increasing its expression would be beneficial for the prevention and therapy of atherosclerosis. In the present study, we studied the effects of ethanolic extracts of Brazilian red propolis (EERP) on ABCA1 expression and cholesterol efflux in THP-1 macrophages. EERP enhanced PPARγ and liver X receptor (LXR) transcriptional activity at 5-15µg/ml, which was associated with upregulation of PPARγ and LXRα expression. It was also found that EERP increase the activity of the ABCA1 promoter, which is positively regulated by LXR. Consistent with these findings, treatment with EERP increased both mRNA and protein expression of ABCA1. Finally, EERP upregulated ApoA-I-mediated cholesterol efflux. Our results showed that EERP promote ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression via induction of PPARγ/LXR.

3,4-Dicaffeoylquinic Acid, a Major Constituent of Brazilian Propolis, Increases TRAIL Expression and Extends the Lifetimes of Mice Infected with the Influenza A Virus.

Brazilian green propolis water extract (PWE) and its chemical components, caffeoylquinic acids, such as 3,4-dicaffeoylquinic acid (3,4-diCQA), act against the influenza A virus (IAV) without influencing the viral components. Here, we evaluated the anti-IAV activities of these compounds in vivo. PWE or PEE (Brazilian green propolis ethanol extract) at a dose of 200 mg/kg was orally administered to Balb/c mice that had been inoculated with IAV strain A/WSN/33. The lifetimes of the PWE-treated mice were significantly extended compared to the untreated mice. Moreover, oral administration of 3,4-diCQA, a constituent of PWE, at a dose of 50 mg/kg had a stronger effect than PWE itself. We found that the amount of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA in the mice that were administered 3,4-diCQA was significantly increased compared to the control group, while H1N1 hemagglutinin (HA) mRNA was slightly decreased. These data indicate that PWE, PEE or 3,4-diCQA possesses a novel and unique mechanism of anti-influenza viral activity, that is, enhancing viral clearance by increasing TRAIL.

Lifespan-extending effects of royal jelly and its related substances on the nematode Caenorhabditis elegans.

BACKGROUND: One of the most important challenges in the study of aging is to discover compounds with longevity-promoting activities and to unravel their underlying mechanisms. Royal jelly (RJ) has been reported to possess diverse beneficial properties. Furthermore, protease-treated RJ (pRJ) has additional pharmacological activities. Exactly how RJ and pRJ exert these effects and which of their components are responsible for these effects are largely unknown. The evolutionarily conserved mechanisms that control longevity have been indicated. The purpose of the present study was to determine whether RJ and its related substances exert a lifespan-extending function in the nematode Caenorhabditis elegans and to gain insights into the active agents in RJ and their mechanism of action. PRINCIPAL FINDINGS: We found that both RJ and pRJ extended the lifespan of C. elegans. The lifespan-extending activity of pRJ was enhanced by Octadecyl-silica column chromatography (pRJ-Fraction 5). pRJ-Fr.5 increased the animals' lifespan in part by acting through the FOXO transcription factor DAF-16, the activation of which is known to promote longevity in C. elegans by reducing insulin/IGF-1 signaling (IIS). pRJ-Fr.5 reduced the expression of ins-9, one of the insulin-like peptide genes. Moreover, pRJ-Fr.5 and reduced IIS shared some common features in terms of their effects on gene expression, such as the up-regulation of dod-3 and the down-regulation of dod-19, dao-4 and fkb-4. 10-Hydroxy-2-decenoic acid (10-HDA), which was present at high concentrations in pRJ-Fr.5, increased lifespan independently of DAF-16 activity. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that RJ and its related substances extend lifespan in C. elegans, suggesting that RJ may contain longevity-promoting factors. Further analysis and characterization of the lifespan-extending agents in RJ and pRJ may broaden our understanding of the gene network involved in longevity regulation in diverse species and may lead to the development of nutraceutical interventions in the aging process.

Caffeoylquinic acids are major constituents with potent anti-influenza effects in brazilian green propolis water extract.

Influenza A viral infections reached pandemic levels in 1918, 1957, 1968, and, most recently, in 2009 with the emergence of the swine-origin H1N1 influenza virus. The development of novel therapeutics or prophylactics for influenza virus infection is urgently needed. We examined the evaluation of the anti-influenza virus (A/WSN/33 (H1N1)) activity of Brazilian green propolis water extract (PWE) and its constituents by cell viability and real-time PCR assays. Our findings showed strong evidence that PWE has an anti-influenza effect and demonstrate that caffeoylquinic acids are the active anti-influenza components of PWE. Furthermore, we have found that the amount of viral RNA per cell remained unchanged even in the presence of PWE, suggesting that PWE has no direct impact on the influenza virus but may have a cytoprotective activity by affecting internal cellular process. These findings indicate that caffeoylquinic acids are the active anti-influenza components of PWE. Above findings might facilitate the prophylactic application of natural products and the realization of novel anti-influenza drugs based on caffeoylquinic acids, as well as further the understanding of cytoprotective intracellular mechanisms in influenza virus-infected cells.

Laxative effects of agarwood on low-fiber diet-induced constipation in rats.

BACKGROUND: Agarwood (Aquilaria sinensis), well known as incense in Southeast Asia, has been used as a digestive in traditional medicine. We investigated the laxative effects of an ethanol extract of agarwood leaves (EEA) in a rat model of low-fiber diet-induced constipation. METHODS: A set of rats was bred on a normal diet while another set was placed on a low-fiber diet to induce constipation. The laxative effect of agarwood was then investigated on both sets of rats. RESULTS: Pretreatment of normal rats with single dose of EEA (600 mg/kg, p.o.) significantly increased frequency and weight of stools. Also, treatments with EEA (300 and 600 mg/kg, p.o.) for 14 days caused a significant increase in stool frequency and weight. Feeding of the animals with a low-fiber diet resulted in a decrease in stool weight, frequency, and water content and also delayed carmine egestion. A single treatment with EEA (600 mg/kg) or senna (150 and 300 mg/kg) significantly increased stool frequency, weight, and water content and also accelerated carmine egestion in the model rats. Once daily administrations of EEA (150 mg/kg), for 14 days, caused a significant increase in water content of stools. The higher doses of EEA (300 and 600 mg/kg) significantly increased frequency, weight, and water content of the stools while accelerating carmine egestion in the constipated rats. Senna (150 and 300 mg/kg) produced similar effect as the higher doses of EEA but, in addition, induced severe diarrhea. CONCLUSION: These findings indicate that EEA has a laxative effect, without causing diarrhea, in a rat model of low-fiber diet-induced constipation. These findings suggest that EEA may be highly effective on constipation as a complementary medicine in humans suffering from life style-induced constipation.

Agarwood induced laxative effects via acetylcholine receptors on loperamide-induced constipation in mice.

Agarwood (Aquilaria sinensis, Aquilaria crasna) is well known as an incense in the oriental region such as Thailand, Taiwan, and Cambodia, and is used as a digestive in traditional medicine. We investigated the laxative effects and mechanism of agarwood leaves extracted with ethanol (EEA-1, Aquilaria sinensis; EEA-2, Aquilaria crasna). EEA-1, EEA-2, the main constituents of EEAs (mangiferin, and genkwanin-5-O-primeveroside), and senna increased the frequency and weight of stools in loperamide-induced constipation model mice. EEA-1 and EEA-2 did not induce diarrhea as a side effect, but senna induced severe diarrhea. EEA-1 and senna increased gastro-intestinal (GI) transit in the model mice. EEA-1, but not senna, also increased the intestinal tension of isolated jejunum and ileum in guinea pigs, and the tension increase was blocked by atropine, a muscarinic receptor antagonist, but not by other inhibitors (granicetron, pyrilamine, or bradykinin-antagonist peptide). Furthermore, the increase in frequency and weight of stools induced by EEA-1 were blocked by pre-administration of atropine in the model mice. These findings indicate that EEAs exerted a laxative effect via acetylcholine receptors in the mouse constipation model.

Anti-inflammatory effect of bee pollen ethanol extract from Cistus sp. of Spanish on carrageenan-induced rat hind paw edema.

BACKGROUND: Bee pollen, a honeybee product, is the feed for honeybees prepared themselves by pollens collecting from plants and has been consumed as a perfect food in Europe, because it is nutritionally well balanced. In this study, we aimed to investigate the anti-inflammatory effect of bee pollen from Cistus sp. of Spanish origin by a method of carrageenan-induced paw edema in rats, and to investigate the mechanism of anti-inflammatory action and also to elucidate components involved in bee pollen extracted with ethanol. METHODS: The bee pollen bulk, its water extract and its ethanol extract were administered orally to rats. One hour later, paw edema was produced by injecting of 1% solution of carrageenan, and paw volume was measured before and after carrageenan injection up to 5 h. The ethanol extract and water extract were measured COX-1 and COX-2 inhibitory activities using COX inhibitor screening assay kit, and were compared for the inhibition of NO production in LPS-stimulated RAW 264.7 cells. The constituents of bee pollen were purified from the ethanol extract subjected to silica gel or LH-20 column chromatography. Each column chromatography fractions were further purified by repeated ODS or silica gel column chromatography. RESULTS: The bee pollen bulk mildly suppressed the carrageenan-induced paw edema and the water extract showed almost no inhibitory activity, but the ethanol extract showed relatively strong inhibition of paw edema. The ethanol extract inhibited the NO production and COX-2 but not COX-1 activity, but the water extract did not affect the NO production or COX activities. Flavonoids were isolated and purified from the ethanol extract of bee pollen, and identified at least five flavonoids and their glycosides. CONCLUSIONS: It is suggested that the ethanol extract of bee pollen show a potent anti-inflammatory activity and its effect acts via the inhibition of NO production, besides the inhibitory activity of COX-2. Some flavonoids included in bee pollen may partly participate in some of the anti-inflammatory action. The bee pollen would be beneficial not only as a dietary supplement but also as a functional food.

Ethanolic extracts of Brazilian red propolis promote adipocyte differentiation through PPARγ activation.

AIM OF THE STUDY: The aim of present study was to investigate the effects of ethanolic extracts of red propolis (EERP) on adipogenesis and evaluate the molecular basis for their anti-obesity effects. MATERIALS AND METHODS: We tested whether EERP alone could induce differentiation of 3T3-L1 cells, regulate the expression of adipocyte-specific genes and reverse inhibitory effects of TNF-α on their differentiation. Next, we performed a luciferase reporter gene assay to test whether EERP could enhance transcriptional activities of PPARγ and adiponectin promoter activities. RESULTS: EERP strongly induced differentiation of 3T3-L1 preadipocytes into adipocytes, and enhanced the PPARγ transcriptional activity and adiponectin promoter activity. In addition, EERP attenuated the inhibitory effect of TNF-α on adipocyte differentiation and adiponectin production in mature adipocytes. CONCLUSION: The present study indicates that EERP enhance differentiation of 3T3-L1 adipocytes in part by its potency of PPARγ activation and are capable of reversing inhibitory effects of TNF-α on adipocyte differentiation and adiponectin expression. These results suggest the value of EERP as a diet supplement for prevention and treatment of obesity and obesity-associated disorders.

Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ), bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs). METHODS: In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE)]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. RESULTS: RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. CONCLUSION: Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.

Antihypertensive effects of flavonoids isolated from brazilian green propolis in spontaneously hypertensive rats.

Propolis, a honeybee product, has become popular as a food and alternative medicine. Its constituents have been shown to exert pharmacological effects, such as anticancer, antimicrobial, and anti-inflammatory effects. The present study was performed to investigate whether Brazilian green propolis exerts antihypertensive effects in spontaneously hypertensive rats (SHR) and which constituents are involved in its effects. Brazilian green propolis was extracted with ethanol and subjected to LH-20 column chromatography eluted with ethanol. The ethanol-eluted fractions at 10 mg/kg were administered orally to SHR for 14 d. Significant decreases in blood pressure were observed in fractions 6 and 7. The active constituents were purified and identified to be four flavonoids: dihydrokaempferide and isosakuranetin in fraction 6 and betuletol and kaempferide in fraction 7. These flavonoids at 10 mg/kg were administered orally to SHR for 28 d, and as a result, isosakuranetin, dihydrokaempferide and betuletol produced significant decrease in blood pressure, especially marked were the effects observed in the group that received isosakuranetin. Brazilian green propolis, fractions 6 and 7, and the 4 active constituents relaxed isolated SHR aorta in a concentration-dependent manner. Therefore, these finding suggest that the vasodilating action may be partly involved in the mechanism of antihypertensive effect. Hence, the ethanol extract of Brazilian green propolis and its main constituents may be useful for prevention of hypertension.

Bilberry and its main constituents have neuroprotective effects against retinal neuronal damage in vitro and in vivo.

Our aim was to determine whether a Vaccinium myrtillus (bilberry) anthocyanoside (VMA) and/or its main anthocyanidin constituents (cyanidin, delphinidin, and malvidin) can protect retinal ganglion cells (RGCs) against retinal damage in vitro and in vivo. In RGC cultures (RGC-5, a rat ganglion cell-line transformed using E1A virus) in vitro, cell damage and radical activation were induced by 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Cell viability was measured using a water-soluble tetrazolium salt assay. Intracellular radical activation within RGC-5 cells was evaluated using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester (CM-H(2)DCFDA). Lipid peroxidation was assessed using the supernatant fraction of mouse forebrain homogenates. In mice in vivo, we evaluated the effects of VMA on N-methyl-D-aspartic acid (NMDA)-induced retinal damage using hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) stainings. VMA and all three anthocyanidins (i) significantly inhibited SIN-1-induced neurotoxicity and radical activation in RGC-5, (ii) concentration-dependently inhibited lipid peroxidation in mouse forebrain homogenates. Intravitreously injected VMA significantly inhibited the NMDA-induced morphological retinal damage and increase in TUNEL-positive cells in the ganglion cell layer. Thus, VMA and its anthocyanidins have neuroprotective effects (exerted at least in part via an anti-oxidation mechanism) in these in vitro and in vivo models of retinal diseases.