Development of Fortified Breads Enriched with Plant-Based Bioactive Peptides Derived from the Chia (Salvia hispanica L.) Expeller.

By-products from the industrialization of oilseeds, particularly chia, can be sustainably used for the development of new functional products. In this work, wheat breads supplemented with up to 10 mg of chia expeller hydrolysate/g of flour were prepared, obtaining fortified breads with acceptability for consumption, according to a preliminary consumer research study based on an affective test employing a five-point hedonic scale of global acceptance. In this context, protein hydrolysates of the chia expeller were produced using Alcalase, reaching a degree of hydrolysis of 54.3 ± 1.6% with an antioxidant activity of 55.8 ± 0.4% after 6 h incubation at 25 °C in the presence of the enzyme. These peptides showed appropriate techno-functional properties and chemical compositions suitable for the further development of bakery products. Taken together, our approach and the development of a fortified bread with plant-based bioactive peptides provide a novel and eco-friendly alternative for the recovery of nutrients from agro-industrial waste. More importantly, these enriched breads could exert beneficial effects on human health by exploiting the antioxidant properties of functional peptides derived from the chia expeller.

Studies on pyridonecarboxylic acids. 1. Synthesis and antibacterial evaluation of 7-substituted-6-halo-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3- carboxylic acids.

A series of [1,3]thiazeto[3,2-a]quinoline-3-carboxylic acids and their esters were prepared and evaluated for antibacterial activity. The derivatives with a hydrogen or methyl group at C-1, fluorine at C-6, and piperazinyl or 4-methyl-1-piperazinyl group at C-7 showed superior in vitro antibacterial activity, and the derivatives with 4-methyl-1-piperazinyl group at C-7 had potent in vivo activity. Compound 29a (NM394) showed excellent in vitro antibacterial activity and low toxicity but poor absorption from the gastrointestinal tract. Compound 29ee (NM441), an N-[(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl] derivative of 29a, was found to possess a favorable pharmacokinetic profile and oral activity superior to that of ciprofloxacin in experimental animals.

Inhibition of stress-stimulated colonic propulsion by alpha 2-adrenoceptor antagonists in rats.

Alpha2-adrenoceptor antagonists have been reported to stimulate colonic motor activity, but the effect on colonic motor dysfunction is unclear. We have investigated the effect of alpha 2-adrenoceptor antagonists on wrap-restraint stress-stimulated and normal colonic propulsion in rats. Colonic propulsion was evaluated by the transit of a charcoal marker along the colon. Faecal pellets output was also measured. A 30-min exposure to wrap-restraint stress starting 120 min after infusion of the charcoal marker significantly stimulated colonic transit with a concomitant increase in faecal pellets. Yohimbine and idazoxan, alpha 2-adrenoceptor antagonists, clonidine, an alpha 2-adrenoceptor agonist, and atropine suppressed wrap-restraint stress-stimulated colonic transit and faecal excretion in a dose-dependent manner. Ondansetron and YM060, 5-hydroxytryptamine3 (5-HT3) receptor antagonists, potently inhibited wrap-restraint stress-stimulated colonic transit, but only weakly inhibited faecal excretion. Neither alpha 2-adrenoceptor antagonists nor atropine had any significant effect on normal colonic transit, whereas clonidine and the 5-HT3 receptor antagonists inhibited it. alpha 2-Adrenoceptor antagonists as well as clonidine, atropine and 5-HT3 receptor antagonists inhibit the stress-induced colonic motor dysfunction in rats.

Nucleoside-nucleotide mixture increases peripheral neutrophils in cyclophosphamide-induced neutropenic mice.

The importance of nucleotides and nucleosides in human nutrition has now become an area of intensive research and clinical interest. To determine any potential benefits of administering a nucleoside-nucleotide mixture (NNM) to cyclophosphamide (CPA)-induced neutropenic mice, we randomly assigned 20 BALB/c mice to two groups and fed them a nucleic-acid-free 20%-casein diet for 20 days. The mice were intraperitoneally administered NNM or saline daily from the onset of the experiment. On the 10th day of this treatment, the mice were intraperitoneally injected with CPA. Blood was collected retro-orbitally and differential counts of leucocytes were done on blood smears by counting microscopically on day 0 (before) and 1, 3, 5, 7, and 10 days after CPA injection. By the 7th and 10th days after CPA injection, respectively, the peripheral neutrophil number in the saline group was significantly lower compared with the neutrophil number in the NNM group (p < 0.05). Also in the NNM group, more clusters of matured neutrophils were observed compared with the saline group. These results suggest that NNM may stimulate the proliferation, differentiation, and maturation of neutrophils, and that NNM alone or in combination with other pharmacologic agents may be an important therapeutic agent in patients after chemotherapy, immunosuppressive therapy, and organ transplant.

Involvement of cholinergic and GABAergic systems in the reversal of memory disruption by NS-105, a cognition enhancer.

The effects of (+)-5-oxo-D-prolinepiperidinamide monohydrate (NS-105) on the scopolamine-, electrolytic lesion of the nucleus basalis magnocellularis (NBM)-, AF64A-, baclofen-, cerebral ischemia- and electroconvulsive shock (ECS)-induced memory disruption in the passive avoidance response or radial arm maze tasks were investigated in rats. The effects of NS-105 were compared with those of aniracetam, bifemelane, idebenone, and indeloxazine in two tasks of the passive avoidance response. Furthermore, effects of NS-105 on in vivo release of acetylcholine (ACh) in the cerebral cortex, high-affinity choline uptake (HACU) of the cerebral cortex in rats with lesion of NBM, HACU of the hippocampus in rats treated with pentobarbital and activity of choline acetyltransferase (ChAT) of the cerebral cortex in rats with lesion of NBM were examined. NS-105 showed antiamnestic actions in a variety of animal models of cholinergic dysfunction employed in this study. Aniracetam improved memory disruption caused by scopolamine, but bifemelane, idebenone, and indeloxazine did not. NS-105 (10 mg/kg) showed the increase of ACh release from the cerebral cortex and the enhancement of HACU both in the cerebral cortex and hippocampus, but showed no change in activity of ChAT. NS-105 also reversed memory disruption induced by baclofen, a potent GABA(B) receptor agonist, but all of reference drugs did not. These results suggest that antiamnestic action of NS-105 is due to the facilitation of cholinergic neuronal activity and the suppression of GABA(B) receptor-mediated responses.

[A stick type filter unit for prevention of biohazard in a research laboratory].

It is important to prevent unexpected laboratory infections in a biosafety area. It is recommended that extreme caution should be used when handling needles and syringes to avoid autoinoculation, and that needles and syringes should be used only when there is no alternative. To remove cells and cell debris, a disk-type microfilter unit, which is recommended using a needle, has been used for filtrating supernatant from HIV-1-infected cell cultures. We compared a stick-type microfilter unit, which can be used without a needle, with a disk-type one for their safety and recovery of human immunodeficiency virus particle after filtration. To confirm the right of our comparison, we observed the filter electron microscopically for the virus particles and virus suspensions were carried out and discussed. In conclusion, the stick-type filter should be improved, since the filter unit is inferior to the disk-type filter unit in purification of the virus, but for the safety is superior to the disk-type.

Intraperitoneal administration of leukotriene B4 (LTB4) and omega-guanidino caproic acid methane sulfonate (GCA) increased the survival of mice challenged with methicillin-resistant Staphylococcus aureus (MRSA).

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) very often complicate management of immunocompromised patients. We studied the effect of leukotriene B4 (LTB4) and epsilon-guanidino caproic acid methane sulfonate (GCA), on MRSA infection. Mice fed a 20% casein diet were intraperitoneally administered LTB4, GCA, or saline (control) daily for 30 days. On the 10th day of this treatment, mice were challenged with MRSA. The survival rate in the control group (20%) was significantly lower than the rates in the GCA (60%) and LTB4 (50%) groups, respectively (p < 0.05). There was a significant reduction of MRSA in the spleen and kidney of the survived mice in GCA group as against mice in the LTB4 and saline groups, indicating a better recovery in GCA group than the other groups. The results suggest that intraperitoneal administration of GCA and LTB4 may play a role in host defense mechanism during MRSA infections.

In vivo antibacterial activity of a prodrug of NM394, a thiazetoquinoline carboxylic acid derivative.

NM394 (6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1,3]-thiazeto [3,2-a]quinoline-3-carboxylic acid) has potent, broad-spectrum antibacterial activity in vitro, but not in vivo. To increase the bioavailability of NM394, various prodrugs were synthesized and tested. One of them, NM441, an N-(5-methyl-2-oxo-1,3-dioxol-4-yl) derivative, showed potent in vivo antibacterial activity. Using thin-layer chromatography-bioautography, we confirmed that after oral administration, NM441 was readily absorbed and hydrolyzed to NM394. Other prodrugs of NM394 were only partially metabolized to NM394. In pharmacokinetic studies in mice and monkeys, we found that the blood levels of NM394 were 7.8 and 5.9 times greater, respectively, when NM441 rather than NM394 was administered. These findings suggest that NM441 is an effective prodrug of NM394.

Synthesis and antibacterial activity of novel 7-substituted-6-fluoro-1- fluoromethyl-4-oxo-4H-[1,3thiazeto[3,2-a]quinoline-3-carboxylic acid derivatives.

A series of 7-substituted-6-fluoro-1-fluoromethyl-4-oxo-4H- [1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid derivatives (2a-1) was prepared and evaluated for antibacterial activity. These compounds were obtained by deacylation of 4-benzoyloxy-2-(1-chloro-2-fluoroethyl)thio-6,7- difluoroquinoline-3-carboxylate (10) and subsequent intramolecular cyclization followed by substitution with cyclic amines and then hydrolysis. The intramolecular cyclization reaction of 18, one of the diastereomers (17, 18) revealed that the cyclization reaction proceeded through an inversion to afford (-)-11a in good chemical and optical yield. The enantiomers of 2a were prepared from the enantiomers of 11a, which were obtained by the optical resolution of the racemate using high-performance liquid chromatography (HPLC). Compounds 2a,b showed excellent in vitro and in vivo antibacterial activity against both gram-negative and gram-positive bacteria including quinolone and Methicillin-resistant Staphylococcus aureus.

New hypocholesterolemic abietamide derivatives. II. Synthesis and hypocholesterolemic activity of N-phenyl-delta 8-dihydroabietamides.

A series of N-phenyl-delta 8-dihydroabietamide analogs were prepared and tested for hypocholesterolemic activity. The effects of substituents of the phenyl moiety on the activities were quantitatively analyzed by using various substituent parameters. The activities were enhanced by the electron-donating effect of ortho and para substitutents and the bulkiness of ortho substituents. A combination of 2,6-dimethylaniline with resin acids other than delta 8-dihydroabietic acid produced lower activities than N-(2,6-dimethylphenyl)-delta 8-dihydroabietamide, abietane-type carboxamides being somewhat stronger than pimarane-type carboxamides. The conversion of the carboxamide group to other groups resulted in more or less of a decrease in activity, giving evidence that the carboxamide group is important to hypocholesterolemic activity.

Studies on pyridonecarboxylic acids. IV. Synthesis and antibacterial activity evaluation of S-(-)- and R-(+)-6-fluoro-1-methyl-4-oxo-7- (1-piperazinyl)-4H-[1,3] thiazeto[3,2-a]quinoline-3-carboxylic acids.

Optically active isomers of 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H- [1,3] thiazeto [3,2-alpha] quinoline-3- carboxylic acid (NM394, 3) were prepared through optical resolution of their racemic intermediate ( +/- )-1 by high-performance liquid chromatography (HPLC). The absolute configuration at the C-1 position in the thiazeto-quinolone ring of ( - )-3 was confirmed by X-ray analysis ( - )-4 to be S. The in vitro antibacterial activity of ( - )-3 was 2--8 times that of (+)-3.

Structure-antibacterial activity and cytotoxicity relationships of thiazolo and thiazetoquinolone derivatives.

In the course of our search for derivatives with potent antibacterial activity and low cytotoxicity, we have studied the relationships among the structure, antibacterial activity and cytotoxicity of thiazoloquinolone and thiazetoquinolone derivatives (the term quinolone as used in this paper includes the quinolone, 1,8-naphthyridine and pyridopyrimidine nuclei). The antibacterial activities and cytotoxicity of these derivatives were compared with those of norfloxacin, ofloxacin, enoxacin and ciprofloxacin. The antibacterial activities of the thiazoloquinolone derivatives were more potent than those of the dihyrothiazoloquinolone derivatives, and comparable to that of ciprofloxacin. All of the thiazoloquinolone derivatives were highly cytotoxic against mammalian cells, but some of the dihyrothiazoloquinolone derivatives were less cytotoxic, being comparable in cytotoxicity to the reference drugs. The thiazetoquinolone derivatives were less cytotoxic than the thiazoloquinolone derivatives, and one of them, 6-fluoro-1-methyl-4-oxo-7-(1-piperazinyl)-4H-[1,3]thiazeto[3,2-a] quinoline-3-carboxylic acid, showed the most potent antibacterial activity of all compounds tested in this study, as well as a very low cytotoxicity. The antibacterial activity and cytotoxicity of this compound were similar to that of ciprofloxacin.

A mixture of nucleosides and nucleotides increases bone marrow cell and peripheral neutrophil number in mice infected with methicillin-resistant Staphylococcus aureus.

We studied the effects of a mixture of nucleosides and nucleotides on the peripheral neutrophil number and the proliferation of bone marrow cells in mice challenged with methicillin-resistant Staphylococcus aureus. BALB/c mice were fed a nucleotide-free 20% casein diet (control) or this diet supplemented with nucleosides and nucleotides orally (Expt. 1) or intraperitoneally (Expt. 2 and 3). On d 10, the mice were challenged intravenously with methicillin-resistant S. aureus (6.7 x 10(12) colony forming units/L). In Expt. 1 and 2, numbers of total and differential counts of blood leucocytes were counted on d 0 (before), 1, 3 and 5 after the infection. In Expt. 3, 30 min before killing, bromodeoxyuridine (20 mg/kg), an analogue of thymidine, was administered intraperitoneally and its incorporation in the DNA synthetic phase of bone marrow cells was determined at 0 h (before), 3, 6 and 24 h after the infection. Mice fed the supplemented diet had higher (P < 0.05) leucocyte and neutrophil numbers on d 0 compared with the control group. The neutrophil numbers tended to be greater in the supplemented group at 1, 3 and 5 d after the infection. Intraperitoneal administration of nucleosides and nucleotides increased (P < 0.05) neutrophil numbers before and after the infection. Twenty-four h after the infection, incorporation of bromodeoxyuridine into the DNA synthetic phase of bone marrow cells in the group administered nucleosides and nucleotides was higher (P < 0.05) than in the control group. We conclude that, following methicillin-resistant S. aureus injection, intraperitoneal administration of a nucleoside-nucleotide mixture may stimulate bone marrow cell proliferation and increase the peripheral blood neutrophil numbers. Oral administration may elicit weaker effects.

Effect of YNS-15P, a new alpha-2 adrenoceptor antagonist, on stress-stimulated colonic propulsion in rats.

We studied effects of a novel alpha-2 adrenoceptor antagonist, YNS-15P (N-[(2R,11bS)-9-methoxy-1,3,4,6,7, 11b-hexahydro-2H-benzoquinolizin-2-yl]-N-methylmethanesulfonami de hydrochloride), on colonic propulsion stimulated by wrap-restraint stress (WRS) or bethanechol, on normal colonic propulsion and on diarrhea induced by castor oil in rats. Alpha-2 adrenoceptor antagonists, rauwolscine and RX821002, decreased the increase in the number and weight of fecal pellets induced by WRS. YNS-15P also inhibited WRS-stimulated fecal excretion in a dose-dependent manner. A 5-hydroxytryptamine3 receptor antagonist, granisetron, trimebutine and diazepam, but not a 5-hydroxytryptamine4 receptor antagonist, GR113808, significantly inhibited WRS-stimulated fecal excretion. YNS-15P inhibited WRS-stimulated colonic transit in a dose-dependent manner. However, YNS-15P had no significant effect on normal fecal excretion and colonic transit or on bethanechol-stimulated fecal excretion. YNS-15P also failed to inhibit castor-oil-induced diarrhea. These results indicate that YNS-15P selectively inhibits WRS-stimulated colonic propulsion, and that alpha-2 adrenoceptors may be involved in stress-induced colonic motor dysfunction in fed rats.

Studies on pyridonecarboxylic acids. III. Synthesis and antibacterial activity evaluation of 1,8-disubstituted 6-fluoro-4-oxo-7-piperazinyl-4H-[1,3]thiazeto[3,2- a]quinoline-3-carboxylic acid derivatives.

A series of 1,8-disubstituted 6-fluoro-4-oxo-7-piperazinyl-4H-[1,3]thiazeto[3,2-a]quinoline-3- carboxylic acid derivatives was prepared and evaluated for antibacterial activity. In the 7-piperazinyl series, addition of a fluorine at C-8, which increased the in vitro activity for the 1-hydrogen and 1-methyl analogues and decreased it for the 1-phenyl analogue, improved the in vivo activity of all the analogues. Introduction of a methoxy group at C-8 of the 1-methyl-7-piperazinyl analogue also improved its in vivo antibacterial activity. The effect of 8-substituents on the in vitro and in vivo antibacterial activity of the 1-methyl-7-(4-methyl-1-piperazinyl) series is also discussed.

In vitro antibacterial activity of a new quinolone, NM394.

NM394 is a new 6-fluoroquinolone antibacterial agent with a tricyclic structure which has a bridge that connects the N-1 and C-2 positions of the quinolone. The antibacterial activity of NM394 against clinical isolates of staphylococci, streptococci, enterococci, members of the family Enterobacteriaceae, and Pseudomonas aeruginosa was equal to or one-half that of ciprofloxacin. NM394 was as active as ofloxacin against gram-positive bacteria and was two to eight times more active against gram-negative bacteria, including P. aeruginosa. NM394 was two to eight times more active than enoxacin against gram-positive and gram-negative bacteria. The MICs of NM394 against Escherichia coli and P. aeruginosa at pH 5.5 were reduced 4 to 16 times compared with those at pH 7.0. Ciprofloxacin, ofloxacin, and enoxacin were 2 to 32 times less active against these two bacteria and Staphylococcus aureus at an acidic pH than they were at pH 7.0. In the presence of 5 mM Mg2+, the MICs of all of these drugs increased 2 to 32 times, but they were only slightly affected by 5 mM Ca2+, type of medium, serum, or size of inoculum. NM394 showed potent bactericidal activity and inhibited the supercoiling activity of E. coli DNA gyrase. The in vitro antibacterial profile of NM394 is similar to that of other 6-fluoroquinolones.

In vivo evaluation of NM441, a new thiazeto-quinoline derivative.

NM441 is a lipophilic prodrug of a new thiazeto-quinoline carboxylic acid derivative NM394, and when it is administered orally it is readily absorbed and hydrolyzed to its parent compound. After oral administration of NM441 at a dose of 20 mg/kg to dogs, the peak concentration of NM394 in plasma was 2.39 micrograms/ml, whereas it was 0.63 micrograms/ml for NM394 administered alone. The in vivo activity of NM441 was compared with those of ciprofloxacin, ofloxacin, and enoxacin in mouse protection studies. NM441 was as effective as ofloxacin and was twice as effective as ciprofloxacin against systemic infection with Staphylococcus aureus. Against infections with streptococci, NM441 was two to three times as effective as ofloxacin and five times as effective as ciprofloxacin. Against infection with Escherichia coli, NM441 was as effective as ciprofloxacin and ofloxacin, but against infections with Klebsiella pneumoniae, Serratia marcescens, and Pseudomonas aeruginosa, NM441 was two to four times as effective as ciprofloxacin and ofloxacin. NM441 was three to seven times as effective as enoxacin in systemic infections. Against urinary tract infections with E. coli, NM441 reduced the number of bacterial CFU per gram of kidney by 1 to 2 log10 more and, with P. aeruginosa, by 1 to 6 log10 more than did ciprofloxacin and ofloxacin. Against respiratory tract infections with K. pneumoniae, NM441 was as effective as ofloxacin and was twice as effective as ciprofloxacin.