Functional compounds in fermented buckwheat sprouts

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.     

Functional Compounds in Fermented Buckwheat Sprouts

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.     

Latent production of angiotensin I-converting enzyme inhibitors from buckwheat protein.

The latent production of angiotensin I-converting enzyme (ACE) Inhibitors from tartary buckwheat (BW) was investigated, and the peptides responsible for ACE inhibition characterized. Intact buckwheat was found to exhibit ACE inhibitory activity having an IC50 value of 3.0 mg/ml. The activity of the protein fraction (IC50: 0.36 mg protein/ml) was not enhanced by pepsin treatment. Pepsin, followed by chymotrypsin and trypsin hydrolysis, resulted in a significant increase in the ACE inhibitory activity (IC50: 0.14 mg protein/ml). The rutin contained in the buckwheat did not exhibit any ACE inhibition. A single oral administration of BW digest lowered the systolic blood pressure of a spontaneously hypertensive rat. Thus, BW proteins offer a potential resource for producing ACE inhibitory peptides during the digestion process. From the di-/tri-peptide fraction (DTPF) of the BW digest, inhibitory peptides were identified. The magnitude (%) of the total ACE inhibitory contribution of each identified peptide, relative to the overall inhibition of the DTPF, was about 41%.     

Direct Resolution of dl-Lysine-3, 5-Dimtrobenzoate

The inhibitory activity of an angiotensin I-converting enzyme (ACE) detected in soy sauce was fractionated into two major fractions of high molecular weight (Hw) and low molecular weight (Lw) by gel filtration chromatography on Bio-gel P-2 after treating with ethanol. The Hw fraction reduced the blood pressure in hypertensive rats after orally administering, while the Lw fraction did not. The ACE inhibitor in the Hw fraction was further purified by Dowex 50W ion-exchange chromatography and four subsequent steps of HPLC. On the basis of the SIMS-mass spectrum, NMR spectrum and other characteristics, the purified ACE inhibitor was identified as nicotianamine (N-[N-(3-amino-3-carboxypropyl)-3-amino-3- carboxypropyl]azetidine-2-carboxylic acid). The IC₅₀ value for this ACE was 0.26 µM.     

An anthocyanin compound in buckwheat sprouts and its contribution to antioxidant capacity

The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.     

ACE/ACE2 Ratio and MMP-9 Activity as Potential Biomarkers in Tuberculous Pleural Effusions

Objective: Pleural effusion is common problem, but the rapid and reliable diagnosis for specific pathogenic effusions are lacking. This study aimed to identify the diagnosis based on clinical variables to differentiate pleural tuberculous exudates from other pleural effusions. We also investigated the role of renin-angiotensin system (RAS) and matrix metalloproteinase (MMPs) in the pathogenesis of pleural exudates.Experimental design: The major components in RAS and extracellular matrix metabolism, including angiotensin converting enzyme (ACE), ACE2, MMP-2 and MMP-9 activities, were measured and compared in the patients with transudative (n = 45) and exudative (n = 80) effusions. The exudative effusions were come from the patients with tuberculosis (n = 20), pneumonia (n = 32), and adenocarcinoma (n = 28).Results: Increased ACE and equivalent ACE2 activities, resulting in a significantly increased ACE/ACE2 ratio in exudates, were detected compared to these values in transudates. MMP-9 activity in exudates was significantly higher than that in transudates. The significant correlation between ACE and ACE2 activity that was found in transudates was not found in exudates. Advanced analyses showed significantly increased ACE and MMP-9 activities, and decreased ACE2 activity in tuberculous pleural effusions compared with those in pneumonia and adenocarcinoma effusions. The results indicate that increased ACE and MMP-9 activities found in the exudates were mainly contributed from a higher level of both enzyme activities in the tuberculous pleural effusions.Conclusion: Interplay between ACE and ACE2, essential functions in the RAS, and abnormal regulation of MMP-9 probably play a pivotal role in the development of exudative effusions. Moreover, the ACE/ACE2 ratio combined with MMP-9 activity in pleural fluid may be potential biomarkers for diagnosing tuberculous pleurisy.     

UV-induced generation of rare tautomers of allopurinol and 9-methylhypoxanthine -- a matrix isolation FTIR study

Monomers of allopurinol and 9-methylhypoxanthine were studied using the matrix isolation technique combined with Fourier transform infrared spectroscopy. The oxo tautomeric forms of both compounds were found to dominate in freshly deposited low-temperature argon matrices. For 9-methylhypoxanthine, a small amount of the hydroxy tautomer was also detected in an Ar matrix before any irradiation. Upon exposure of the matrices to the UV (lambda>230 nm or lambda>270 nm) light, a proton transfer photoreaction converting the oxo forms of both compounds into the corresponding hydroxy tautomers occurred. Generation of conjugated ketenes as minor photoproducts was also observed. For 4(3H)-pyrimidinone (a model compound for both allopurinol and 9-methylhypoxanthine), photoreversibility of the UV-induced oxo --> hydroxy transformation was experimentally proven by direct observation of the back hydroxy --> oxo photoreaction. The substrates (oxo tautomers) and products (hydroxy tautomers) of the observed phototransformations were identified by comparison of their IR spectra with the spectra theoretically predicted at the DFT(B3LYP)/6-31++G(d,p) level. The IR bands in the experimental spectra were assigned to the calculated normal modes.     

Cytotoxic Alangium alkaloids from Alangium longiflorum

Seven alkaloids (1-7) were isolated from the stem bark of Alangium longiflorum. Compound 1, (-)-10-O-demethylisocephaeline, was isolated for the first time as a naturally occurring product from a plant source. All structures were elucidated by detailed spectroscopic analysis. Biological evaluation showed that 2, 10-O-demethylcephaeline, exhibited potent cytotoxic activity against human lung carcinoma (A549) and breast adenocarcinoma (MCF-7) with ED(50) values of 0.013 and 0.062 microM, respectively. The stereoisomer 1 was less potent than 2, and related compounds with different hydroxy/methoxy substitution patterns were also less potent or inactive. Thus, compound 2 merits attention as a cytotoxic lead for further study.     

Microbial Oxidation of sec-Alcohols to Ketones

The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.     

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 μM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 μM DX600 (>95% inhibition) but not with 1 μM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.     

Design, synthesis, and pharmacological evaluation of N-bicyclo-5-chloro-1H-indole-2-carboxamide derivatives as potent glycogen phosphorylase inhibitors

As a result of the various N-bicyclo-5-chloro-1H-indole-2-carboxamide derivatives with a hydroxy moiety synthesized in an effort to discover novel glycogen phosphorylase (GP) inhibitors, 5-chloro-N-(5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-1H-indole-2-carboxamide (5b) was found to have potent inhibitory activity. The introduction of fluorine atoms both at a position adjacent to the hydroxy group and in the central benzene moiety lead to the optically active derivative 5-chloro-N-[(5R)-1,3,6,6-tetrafluoro-5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl]-1H-indole-2-carboxamide (25e(alpha), which was the most potent compound in this series (IC(50)=0.020microM). This compound inhibited glucagon-induced glucose output in cultured primary hepatocytes with an IC(50) value of 0.69microM, and showed oral hypoglycemic activity in diabetic db/db mice at 10mg/kg. Compound 25e(alpha) also had an excellent pharmacokinetic profile, with high oral bioavailability and a long plasma half-life, in male SD rats. The binding mode of 25e(alpha) to this molecule and the role of fluorine atoms in that binding were speculated in an enzyme docking study.     

The role of nicotianamine synthase in response to Fe nutrition status in Gramineae

Nicotianamine is an intermediate for the biosynthesis of mugineic acid-family phytosiderophores (MAs) in the Gramineae and a key substance for iron metabolism in dicots. Nicotianamine synthase catalyzes the formation of nicotianamine from S-adenosylmethionine. Nicotianamine synthase activity was induced in barley roots at the 3rd day after withholding Fe supply and declined within one day followmg the supply of Fe³⁺-epihydroxymugineic acid. The induction of nicotianamine synthase activity by Fe-deficiency was observed also in sorghum, maize, and rye, and the level of nicotianamine synthase activity was highly associated with the MAs secreted among graminaceous plant tested. Therefore, the nicotianamine synthase gene may be a suitable candidate for making a transgenic plant tolerant to Fe-deficiency.Abbreviations p-APMSF (p-amidinophenyl) methanesulfonylfluoride hydrochloride - NA nicotianamine - DMA 2-deoxymugineic acid - E-64 trans-epoxysuccinyl-leucylamido-(4-guanidino) butane - epiHMA 3-epihydroxymugineic acid - MAs mugineic acid-family phytosiderophores which include deoxymugineic acid, mugineic acid, hydroxymugineic acid, epihydroxymugineic acid and avenic acid - PVP polyvinylpyrrolidone - SAM S-adenosylmethionine     

A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading

The Arabidopsis Yellow Stripe 1-Like (YSL) proteins have been identified by homology with the maize (Zea mays) Yellow Stripe 1 (YS1) transporter which is responsible for iron-phytosiderophore (PS) uptake by roots in response to iron shortage. Although dicotyledonous plants do not synthesize PS, they do synthesize the PS precursor nicotianamine, a strong metal chelator essential for maintenance of iron homeostasis and copper translocation. Furthermore, ZmYS1 and the rice (Oryza sativa) protein OsYSL2 have metal-nicotianamine transport activities in heterologous expression systems. In this work, we have characterized the function of AtYSL1 in planta. Two insertional loss-of-function ysl1 mutants of Arabidopsis were found to exhibit increased nicotianamine accumulation in shoots. More importantly, seeds of both ysl1 knockouts contained less iron and nicotianamine than wild-type seeds, even when produced by plants grown in the presence of an excess of iron. This phenotype could be reverted by expressing the wild-type AtYSL1 gene in ysl1 plants. ysl1 seeds germinated slowly, but this defect was rescued by an iron supply. AtYSL1 was expressed in the xylem parenchyma of leaves, where it was upregulated in response to iron excess, as well as in pollen and in young silique parts. This pattern is consistent with long-distance circulation of iron and nicotianamine and their delivery to the seed. Taken together, our work provides strong physiological evidence that iron and nicotianamine levels in seeds rely in part on AtYSL1 function.     

Trypanosoma cruzi: antiprotozoal activity of parthenolide obtained from Tanacetum parthenium (L.) Schultz Bip. (Asteraceae, Compositae) against epimastigote and amastigote forms

This study reports the activity of crude extracts, fractions and parthenolide (pure compound) obtained from Tanacetum parthenium against two forms of the parasite Trypanosoma cruzi. Feverfew is a traditional herbal medicine that has been used for the treatment of migraine, fever and arthritis. Activity against epimastigote forms was observed for crude extracts, fractions and parthenolide, and a progressive increase in the antitrypanosomal effect was observed in the course of the purification process. The pure compound showed IC50/96h and IC90/96h of 0.5 microg/ml and 1.25 microg/ml, respectively. The cytotoxic effect of parthenolide in LLMCK2 cells was 3.2 microg/ml (CC50/96h) and the selectivity index was 6.4. No hemolysis was detected for the pure compound. The internalization index of T. cruzi in LLMCK2 cells was reduced almost 51% at the concentration of 2 microg/ml of parthenolide, and 96.6% at 4 microg/ml. Scanning and transmission electron microscopy permitted observation of morphological modifications and ultrastructural alterations.     

荞麦与大豆叶片中草酸含量差异及其可能的原因

用1/5浓度Hoagland营养液培养荞麦和大豆幼苗l0 d后,荞麦叶片、根及其分泌物中的草酸含量均明显高于大豆,说明荞麦叶片的草酸形成能力强.荞麦叶片中存在少量的草酸氧化酶活性,而大豆中未检测到该酶活性,表明荞麦具有一定的降解草酸的能力.乙醇酸氧化酶(GO)催化乙醇酸氧化的活性两种植物之间虽差异不明显,但该酶催化乙醛酸氧化的活性荞麦显著高于大豆.荞麦GO对乙醛酸的Km值明显低于大豆GO,同时其乙醛酸含量也较高,因此其叶片中由乙醛酸形成草酸的速率应高于大豆.由此认为,由乙醛酸氧化生成草酸可能是植物草酸合成限速步骤之一,其反应速率高低可能导致不同种类植物叶片中草酸含量的差异.     

Pulmonary AngII promotes LPS-induced lung inflammation by regulating microRNA-143

Angiotensin converting enzyme 2 (ACE2) is a terminal carboxypeptidase, which cleaves single terminal residues from several bioactive peptides such as Angiotensin II (AngII). Many investigations indicated that ACE2 functions in angiotensin system and plays a crucial role in inflammatory lung diseases. However, the mechanism behind the involvement of ACE2 in inflammatory lung disease has not been fully elucidated. In this study, BEAS-2B cells were treated with gradient concentration of AngII and lipopolysaccharide (LPS) to induce inflammatory condition. Quantitative RT-PCR was performed to detect the level of ACE2 and miR-143-3p. Western blotting and immunofluorescence assays were performed to measure the expression of related proteins. The levels of inflammatory cytokines and cell viability were respectively measured by ELISA and CCK-8 kits. And ACE2 activity was detected by corresponding commercial kits. Bioinformatics methods were employed to predict the potential microRNA targeting ACE2, which was then confirmed by dual luciferase reporter assay. The results showed that ACE2 expression and activity were time-dependently decreased in LPS group for the first 12 h, after which this tendency was reversed. AngII addition enhanced these effects, compared with LPS group. Additionally, the lowest ACE2 activity level was found in both LPS and AngII + LPS groups at 6 h. The number of nuclei and the ACE2 expression were decreased in LPS groups at 6 h and further reduced by addition of AngII, detected by immunofluorescence. Moreover, ACE2 was validated to be a direct target of miR-143-3p. Pretreatment of AngII and LPS regulated the activity of ACE2, increased the expression of proinflammatory cytokines and cell apoptosis and regulated the expression of Bax, Bcl-2 and cleaved caspase-3 in BEAS-2B cells, which could be reversed by transfecting miR-143-3p inhibitor. The results collectively suggest that AngII promotes LPS-induced inflammation by regulating miR-143-3p in BEAS-2B cells. Therefore, miR-143-3p is considered a potential molecular target for the treatment of lung inflammation.     

Isolation and antimicrobial evaluation of isomeric hydroxy ketones in leaf cuticular waxes of Annona squamosa

A novel natural compound, 11-hydroxy-16-hentriacontanone, has been isolated from the leaf cuticular wax of Annona squamosa along with its known isomer 10-hydroxy-16-hentriacontanone in a ratio of 67:33. This isomeric mixture of hydroxy ketones constituted together 16.5% of the total cuticular waxes. The new compound was characterised using spectral and chromatographic techniques. The major component was found to be 16-hentriacontanone (palmitone), which constituted up to 48% of the total cuticular wax, together with a homologous series of hydrocarbons, fatty aldehydes, fatty alcohols, fatty acids and sterols as minor components. The antimicrobial activity of the isomeric hydroxy ketones was tested against selected Gram-positive and Gram-negative bacterial strains, and also some selected fungal strains, and compared with palmitone. The antibacterial activity of palmitone was significantly higher than that of the isomeric hydroxy ketones, but their antifungal activities were comparable.     

Microbial transformation of 17alpha-ethynyl- and 17alpha-ethylsteroids, and tyrosinase inhibitory activity of transformed products

The microbial transformation of the 17alpha-ethynyl-17beta-hydroxyandrost-4-en-3-one (1) (ethisterone) and 17alpha-ethyl-17beta-hydroxyandrost-4-en-3-one (2) by the fungi Cephalosporium aphidicola and Cunninghamella elegans were investigated. Incubation of compound 1 with C. aphidicola afforded oxidized derivative, 17alpha-ethynyl-17beta-hydroxyandrosta-1,4-dien-3-one (3), while with C. elegans afforded a new hydroxy derivative, 17alpha-ethynyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (4). On the other hand, the incubation of compound 2 with the fungus C. aphidicola afforded 17alpha-ethyl-17beta-hydroxyandrosta-1,4-dien-3-one (5). Two new hydroxylated derivatives, 17alpha-ethyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (6) and 17alpha-ethyl-6alpha,17beta-dihydroxy-5alpha-androstan-3-one (7) were obtained from the incubation of compound 2 with C. elegans. Compounds 1-6 exhibited tyrosinase inhibitory activity, with compound 6 being the most potent member (IC(50)=1.72 microM).     

Increased urinary angiotensin-converting enzyme 2 in renal transplant patients with diabetes

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be a renoprotective enzyme, since it converts angiotensin (Ang) II to Ang-(1-7). ACE2 has been detected in urine from patients with chronic kidney disease. We measured urinary ACE2 activity and protein levels in renal transplant patients (age 54 yrs, 65% male, 38% diabetes, n?=?100) and healthy controls (age 45 yrs, 26% male, n?=?50), and determined factors associated with elevated urinary ACE2 in the patients. Urine from transplant subjects was also assayed for ACE mRNA and protein. No subjects were taking inhibitors of the renin-angiotensin system. Urinary ACE2 levels were significantly higher in transplant patients compared to controls (p?=?0.003 for ACE2 activity, and p≤0.001 for ACE2 protein by ELISA or western analysis). Transplant patients with diabetes mellitus had significantly increased urinary ACE2 activity and protein levels compared to non-diabetics (p<0.001), while ACE2 mRNA levels did not differ. Urinary ACE activity and protein were significantly increased in diabetic transplant subjects, while ACE mRNA levels did not differ from non-diabetic subjects. After adjusting for confounding variables, diabetes was significantly associated with urinary ACE2 activity (p?=?0.003) and protein levels (p<0.001), while female gender was associated with urinary mRNA levels for both ACE2 and ACE. These data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells. Urinary ACE2 could be a marker of renal renin-angiotensin system activation in these patients.     

Alternative Roles of STAT3 and MAPK Signaling Pathways in the MMPs Activation and Progression of Lung Injury Induced by Cigarette Smoke Exposure in ACE2 Knockout Mice

Inflammation-mediated abnormalities in the renin-angiotensin system (RAS) and expression of matrix metalloproteinases (MMPs) are implicated in the pathogenesis of lung injury. Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that displays antagonist effects on ACE/angiotensin II (Ang II) axis, could also play a protective role against lung diseases. However, the relationship between ACE2 and MMPs activation in lung injury is still largely unclear. The purpose of this study is to investigate whether MMPs activity could be affected by ACE2 and which ACE2 derived signaling pathways could be also involved via using a mouse model with lung injury induced by cigarette smoke (CS) exposure for 1 to 3 weeks. Wild-type (WT; C57BL/6) and ACE2 KO mice (ACE2^-/-) were utilized to study CS-induced lung injury. Increases in the resting respiratory rate (RRR), pulmonary immunokines, leukocyte infiltration and bronchial hyperplasia were observed in the CS-exposed mice. Compared to WT mice, more serious physiopathological changes were found in ACE2^-/- mice in the first week of CS exposure. CS exposure increased pulmonary ACE and ACE2 activities in WT mice, and significantly increased ACE in ACE2^-/- mice. Furthermore, the activity of pulmonary MMPs was decreased in CS-exposed WT mice, whereas this activity was increased in ACE2^-/- mice. CS exposure increased the pulmonary p-p38, p-JNK and p-ERK1/2 level in all mice. In ACE2^-/- mice, a significant increase p-STAT3 signaling was detected; however, no effect was observed on the p-STAT3 level in WT mice. Our results support the hypothesis that ACE2 deficiency influences MMPs activation and STAT3 phosphorylation signaling to promote more pulmonary inflammation in the development of lung injury.