Enhancement of subunit vaccine delivery with zinc-carnosine coordination polymer through the addition of mannan.

Vaccines represent a pivotal health advancement for preventing infection. However, because carrier systems with repeated administration can invoke carrier-targeted immune responses that diminish subsequent immune responses (e.g., PEG antibodies), there is a continual need to develop novel vaccine platforms. Zinc carnosine microparticles (ZnCar MPs), which are composed of a one-dimensional coordination polymer formed between carnosine and the metal ion zinc, have exhibited efficacy in inducing an immune response against influenza. However, ZnCar MPs' limited suspendability hinders clinical application. In this study, we address this issue by mixing mannan, a polysaccharide derived from yeast, with ZnCar MPs. We show that the addition of mannan increases the suspendability of this promising vaccine formulation. Additionally, since mannan is an adjuvant, we illustrate that the addition of mannan increases the antibody response and T cell response when mixed with ZnCar MPs. Mice vaccinated with mannan + OVA/ZnCar MPs had elevated serum IgG and IgG1 levels in comparison to vaccination without mannan. Moreover, in the mannan + OVA/ZnCar MPs vaccinated group, mucosal washes demonstrated increased IgG, IgG1, and IgG2c titers, and antigen recall assays showed enhanced IFN-γ production in response to MHC-I and MHC-II immunodominant peptide restimulation, compared to the vaccination without mannan. These findings suggest that the use of mannan mixed with ZnCar MPs holds potential for subunit vaccination and its improved suspendability further promotes clinical translation.

Whole-cell biotransformation for large scale production of carcinine in Escherichia coli.

Carcinine is a natural imidazole-containing peptide derivative. It is widely used in the cosmetics industry as anti-aging supplement with antioxidant, anti-glycation and glycation reversal functions, and it also has a notable pharmacological effect as anti-tumor drug and in protection against retinopathy. However, a technological method for synthesis and production of carcinine has not been established. In this study, a whole-cell transformation system converting ß-alanine and histamine to carcinine by the enzymes Ebony and phosphopantetheine transferase (Sfp) has been developed. The results revealed that the catalytic efficiency of the strain containing the fusion protein of Ebony and Sfp (Sfp-glycine-serine-glycine-Ebony, SGE) in Escherichia coli W3110 (WSGE strain) is significantly higher (7.45 mM) than the combinatorial strain of pET28a-ebony and pACYCDuet-sfp in E. coli BL21(DE3) (BSE strain) (2.17 mM). Under the optimal reaction conditions (25 â„ƒ, pH 7.0, 12.5 g/L wet cells, 20 mM ß-alanine and 40 mM histamine), the carcinine can be quickly synthesized within 24 h up to a concentration of 22.63 mM. To achieve a continuous and efficient conversion of the precursors, a batch-feeding catalysis was designed. With this system, ß-alanine (40 mM) and histamine (40 mM) could be completely transformed to carcinine (40.34 mM) in 36 h with a productivity of 0.204 g/L h reaching a titer of 7.34 g/L. Hence, the batch-feeding whole-cell biocatalysis is a promising technology for the high yield production of carcinine which can promote the industrial production of carcinine.

Erythrocytes Prevent Degradation of Carnosine by Human Serum Carnosinase.

The naturally occurring dipeptide carnosine (ß-alanyl-l-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography-mass spectrometry. In addition, we studied carnosine's effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase.

Development and validation of a HPLC method for the direct separation of carnosine enantiomers and analogues in dietary supplements.

The chiral purity of some molecules such as nutraceuticals is fundamental to ensure their beneficial activities and it must be checked during quality control analysis. Carnosine is a natural histidine dipeptide used as ingredient for food supplements, but only his L-enantiomer is absorbed and active. Despite of this feature, a method for the separation of carnosine enantiomers without derivatization has only recently been published. Herein, we validated a method based on a Chirobiotic T column and an UV detector for the direct quantification of carnosine enantiomers, following ICH guideline. Moreover, we demonstrated that elution with water containing 0.1% formic acid and 20-40% ensures stereo-, chemo- and regio-selectivity for the separation and the identification of carnosine enantiomers and natural analogues. Moreover, the method allows a direct hyphenation with electrospray mass spectrometry to increase detection selectivity and sensitivity. As far as we know, this is the first method allowing the simultaneous identification and quantification of natural analogues of carnosine, which can be important for application such as the identification of enantiomeric impurities or adulteration that can occur during the storage or the preparation of foods or food supplements containing histidine dipeptides.

Biosynthesis, characterization and cytotoxicity of gold nanoparticles and their loading with N-acetylcarnosine for cataract treatment.

The present work showed the biofabrication and characterization of gold nanoparticles (Au NPs) using Coccinia grandis bark extract. The fabricated NPs were well characterized by using different microscopic an spectroscopic techniques such as transmission electron microscopy (TEM), Ultra violet - visible spectroscopy (UV-Vis), X-ray diffraction (XRD), Energy dispersive spectroscopy (EDS) and Fourier transform spectroscopy (FTIR). TEM results showed that the prepared AuNPs are spherical in shape with uniformity in size. The calculated average size of the AuNPs is 20 nm. The NAC drug molecule that is used for cataract treatment was successfully encapsulated into Au NPs to increase its bioavailability. Also, the in-vitro cytotoxicity of NAC and NAC - Au NPs were studied against fibroblast cells, and the results showed that encapsulation of NAC into Au NPs did not showed cytotoxicity after encapsulation. NAC molecules do not exhibit toxicity at lower concentrations, While, there is a reduction in the number of viable cells at higher concentration of NAC. Also, the encapsulation of the drug onto Au NPs is considerably increased biocompatibility and bioavailability. In future, this research results may be helpful for the development of drugs for treatment of cataract with high stability and reactivity.

ß-alanine supplementation to improve exercise capacity and performance: a systematic review and meta-analysis.

OBJECTIVE: To conduct a systematic review and meta-analysis of the evidence on the effects of ß-alanine supplementation on exercise capacity and performance. DESIGN: This study was designed in accordance with PRISMA guidelines. A 3-level mixed effects model was employed to model effect sizes and account for dependencies within data. DATA SOURCES: 3 databases (PubMed, Google Scholar, Web of Science) were searched using a number of terms ('ß-alanine' and 'Beta-alanine' combined with 'supplementation', 'exercise', 'training', 'athlete', 'performance' and 'carnosine'). ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Inclusion/exclusion criteria limited articles to double-blinded, placebo-controlled studies investigating the effects of ß-alanine supplementation on an exercise measure. All healthy participant populations were considered, while supplementation protocols were restricted to chronic ingestion. Cross-over designs were excluded due to the long washout period for skeletal muscle carnosine following supplementation. A single outcome measure was extracted for each exercise protocol and converted to effect sizes for meta-analyses. RESULTS: 40 individual studies employing 65 different exercise protocols and totalling 70 exercise measures in 1461 participants were included in the analyses. A significant overall effect size of 0.18 (95% CI 0.08 to 0.28) was shown. Meta-regression demonstrated that exercise duration significantly (p=0.004) moderated effect sizes. Subgroup analyses also identified the type of exercise as a significant (p=0.013) moderator of effect sizes within an exercise time frame of 0.5-10 min with greater effect sizes for exercise capacity (0.4998 (95% CI 0.246 to 0.753)) versus performance (0.1078 (95% CI -0.201 to 0.416)). There was no moderating effect of training status (p=0.559), intermittent or continuous exercise (p=0.436) or total amount of ß-alanine ingested (p=0.438). Co-supplementation with sodium bicarbonate resulted in the largest effect size when compared with placebo (0.43 (95% CI 0.22 to 0.64)). SUMMARY/CONCLUSIONS: ß-alanine had a significant overall effect while subgroup analyses revealed a number of modifying factors. These data allow individuals to make informed decisions as to the likelihood of an ergogenic effect with ß-alanine supplementation based on their chosen exercise modality.

Effect of Four Weeks of ß-alanine Supplementation on Muscle Carnosine and Blood Serum Lactate during Exercise in Male Rats.

ß-alanine (BA) supplementation may increase muscle buffering capacity and affect physiological responses during exercise. We examined the effects of 4 weeks of BA supplementation on muscle carnosine and serum lactate in male rats. Rats (n = 24, age: 2 months, body weight: 265±22 g) were divided into a BA supplementation or control group. Along with aerobic acclimatization exercise (15 m·min(-1), 8-10 min·day(-1), 4 days·week(-1) for 4 weeks), the BA group had access to BA powder in their drinking water (1.8%) with the control group having access to plain water for 4 weeks. After 4 weeks, rats ran on a treadmill at speeds of 15, 20, 25, 30, and 35 m·min(-1), respectively, each for 4 min, in order to measure post-exercise serum lactate. Muscle carnosine and serum lactate levels were measured with high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbant assay (ELISA) procedures, respectively. Following BA supplementation, carnosine content in the m.rectus femoris increased by 117% (p < .01) and serum lactate decreased by 7.4% (p < .01). It was concluded that ß-alanine supplementation increases muscle carnosine content and reduces serum lactate; these changes may indicate an adaptation of rat skeletal muscles to postpone peripheral muscle fatigue during high-intensity exercise.

ß-Alanine supplementation and military performance.

During sustained high-intensity military training or simulated combat exercises, significant decreases in physical performance measures are often seen. The use of dietary supplements is becoming increasingly popular among military personnel, with more than half of the US soldiers deployed or garrisoned reported to using dietary supplements. ß-Alanine is a popular supplement used primarily by strength and power athletes to enhance performance, as well as training aimed at improving muscle growth, strength and power. However, there is limited research examining the efficacy of ß-alanine in soldiers conducting operationally relevant tasks. The gains brought about by ß-alanine use by selected competitive athletes appears to be relevant also for certain physiological demands common to military personnel during part of their training program. Medical and health personnel within the military are expected to extrapolate and implement relevant knowledge and doctrine from research performed on other population groups. The evidence supporting the use of ß-alanine in competitive and recreational athletic populations suggests that similar benefits would also be observed among tactical athletes. However, recent studies in military personnel have provided direct evidence supporting the use of ß-alanine supplementation for enhancing combat-specific performance. This appears to be most relevant for high-intensity activities lasting 60-300 s. Further, limited evidence has recently been presented suggesting that ß-alanine supplementation may enhance cognitive function and promote resiliency during highly stressful situations.

ß-Alanine Supplementation Does Not Augment the Skeletal Muscle Adaptive Response to 6 Weeks of Sprint Interval Training.

Sprint interval training (SIT), repeated bouts of high-intensity exercise, improves skeletal muscle oxidative capacity and exercise performance. ß-alanine (ß-ALA) supplementation has been shown to enhance exercise performance, which led us to hypothesize that chronic ß-ALA supplementation would augment work capacity during SIT and augment training-induced adaptations in skeletal muscle and performance. Twenty-four active but untrained men (23 ± 2 yr; VO2peak = 50 ± 6 mL · kg(-1) · min(-1)) ingested 3.2 g/day of ß-ALA or a placebo (PLA) for a total of 10 weeks (n = 12 per group). Following 4 weeks of baseline supplementation, participants completed a 6-week SIT intervention. Each of 3 weekly sessions consisted of 4-6 Wingate tests, i.e., 30-s bouts of maximal cycling, interspersed with 4 min of recovery. Before and after the 6-week SIT program, participants completed a 250-kJ time trial and a repeated sprint test. Biopsies (v. lateralis) revealed that skeletal muscle carnosine content increased by 33% and 52%, respectively, after 4 and 10 weeks of ß-ALA supplementation, but was unchanged in PLA. Total work performed during each training session was similar across treatments. SIT increased markers of mitochondrial content, including cytochome c oxidase (40%) and ß-hydroxyacyl-CoA dehydrogenase maximal activities (19%), as well as VO2peak (9%), repeated-sprint capacity (5%), and 250-kJ time trial performance (13%), but there were no differences between treatments for any measure (p < .01, main effects for time; p > .05, interaction effects). The training stimulus may have overwhelmed any potential influence of ß-ALA, or the supplementation protocol was insufficient to alter the variables to a detectable extent.

An "enigmatic" L-carnosine (ß-alanyl-L-histidine)? Cell proliferative activity as a fundamental property of a natural dipeptide inherent to traditional antioxidant, anti-aging biological activities: balancing and a hormonally correct agent, novel patented oral therapy dosage formulation for mobility, skeletal muscle power and functional performance, hypothalamic-pituitary- brain relationship in health, aging and stress studies.

Hypothalamic releasing and inhibiting hormones are major neuroendocrine regulators of human body metabolism being driven directly to the anterior pituitary gland via hypothalamic-hypophyseal portal veins. The alternative physiological or therapeutic interventions utilizing the pharmaco-nutritional boost of imidazole-containing dipeptides (non-hydrolized oral form of carnosine, carcinine, N-acetylcarnosine lubricant eye drops) can maintain health, enhance physical exercise performance and prevent ageing. Carnosine (ß-alanyl-L-histidine) is synthesized in mammalian skeletal muscle. There is an evidence that the release of carnosine from the skeletal muscle sarcomeres moieties during physical exercise affects autonomic neurotransmission and physiological functions. Carnosine released from skeletal muscle during exercise acts as a powerful afferent physiological signaling stimulus for hypothalamus, may be transported into the hypothalamic tuberomammillary nucleus (TMN), specifically to TMN-histamine neurons and hydrolyzed herewith via activities of carnosine-degrading enzyme (carnosinase 2) localized in situ. Through the colocalized enzymatic activity of Histidine decarboxylase in the histaminergic neurons, the resulting L-histidine may subsequently be converted into histamine, which could be responsible for the effects of carnosine on neurotransmission and physiological function. Carnosine and its imidazole-containing dipeptide derivatives are renowned for their anti-aging, antioxidant, membrane protective, metal ion chelating, buffering, anti-glycation/ transglycating activities used to prevent and treat a spectrum of age-related and metabolic diseases, such as neurodegenerative disease, sight threatening eye diseases, Diabetes mellitus and its complications, cancers and other disorders due to their wide spectrum biological activities. The precursor of carnosine (and related imidazole containing compounds) synthesis in skeletal muscles beta-alanine is used as the oral supplement by athletes to achieve the fine sporting art results due to the buffering activities of carnosine and its related imidazole- containing compounds which contribute to the maintenance of the acid-base balance in the acting muscles. This work originally emphasizes that overall data indicate the signaling activities of carnosine in skeletal and cardiac muscles switching on the mechanisms of exercise-induced telomere protection and point to the stress response and growth/cellular proliferation pathways as high-priority candidates for the ongoing studies and therapeutic concepts. The therapeutic interventions utilizing the specific oral formulation (Can-C Plus), timing dosing and pharmaco-nutritional boost of imidazolecontaining dipeptides can maintain health, enhance physical exercise performance and prevent aging. The patented therapeutic concept protects the existence of the interesting physiological major activities, better controls and therapeutic treatments for aging/age-related disorders (including age-related loss of muscle mass and muscle function) using carnosine dipeptide for cellular rejuvenation and manipulating telomeres and enzyme telomerase activity that may reduce some of the physiological declines that accompany aging.

Enhancing antioxidant activity of sesamol at frying temperature by addition of additives through reducing volatility.

Additives were evaluated to investigate their effects on volatility of sesamol at frying temperature with the hypothesis that the interaction between an additive and sesamol would reduce sesamol volatility. Twenty-two additive : sesamol combinations were examined by thermogravimetric analysis (TGA) under nitrogen in neat form and in soybean oil. The results indicate that these additives could bind to or interact with sesamol and consequently reduced its volatility. (1) H NMR study provided evidence for hydrogen bonding between sesamol and a hydroxyl group, an amino group, and ether groups. Subsequent heating tests were conducted to investigate the effect of the reduced volatility of sesamol on antioxidant activity in soybean oil at 180 °C. Oxidation of soybean oil was monitored with gel permeation chromatography for formation of polymerized triacylglycerols and with (1) H NMR for loss of olefinic and bisallylic protons. Sesamol retained in soybean oil during the heating process was determined by high-performance liquid chromatography. A strong correlation between the retained sesamol and the antioxidant activity was observed. The mixture of 830 ppm sesamol and mono-/diglycerides, polysorbate 20 or l-carnosine showed much improved antioxidant activity compared to sesamol itself and slightly better antioxidant activity than 200 ppm tert-butylhydroquinone. It is believed that this method can also be used for many other antioxidants for which volatility is a problem.

Skin beautification with oral non-hydrolized versions of carnosine and carcinine: Effective therapeutic management and cosmetic skincare solutions against oxidative glycation and free-radical production as a causal mechanism of diabetic complications and skin aging.

Advanced glycation Maillard reaction end products (AGEs) are causing the complications of diabetes and skin aging, primarily via adventitious and cross-linking of proteins. Long-lived proteins such as structural collagen are particularly implicated as pathogenic targets of AGE processes. The formation of α-dicarbonyl compounds represents an important step for cross-linking proteins in the glycation or Maillard reaction. The purpose of this study was to investigate the contribution of glycation coupled to the glycation free-radical oxidation reactions as markers of protein damage in the aging of skin tissue proteins and diabetes. To elucidate the mechanism for the cross-linking reaction, we studied the reaction between a three-carbon α-dicarbonyl compound, methylglyoxal, and amino acids using EPR spectroscopy, a spectrophotometric kinetic assay of superoxide anion production at the site of glycation and a chemiluminescence technique. The transglycating activity, inhibition of transition metal ions peroxidative catalysts, resistance to hydrolysis of carnosine mimetic peptide-based compounds with carnosinase and the protective effects of carnosine, carcinine and related compounds against the oxidative damage of proteins and lipid membranes were assessed in a number of biochemical and model systems. A 4-month randomized, double-blind, controlled study was undertaken including 42 subjects where the oral supplement of non-hydrolized carnosine (Can-C Plus® formulation) was tested against placebo for 3 months followed by a 1-month supplement-free period for both groups to assess lasting effects. Assessment of the age-related skin parameters and oral treatment efficacy measurements included objective skin surface evaluation with Visioscan® VC 98 and visual assessment of skin appearance parameters. The results together confirm that a direct one-electron transfer between a Schiff base methylglyoxal dialkylimine (or its protonated form) and methylglyoxal is responsible for the generation of the cross-linked radical cation and the radical counteranion of methylglyoxal. Under aerobic conditions, molecular oxygen can then accept an electron from the methylglyoxal anion to generate the superoxide radical anion causing the propagation of oxidative stress chain reactions in the presence of transition metal ions. Carnosine stabilized from enzymatic hydrolysis, carcinine and leucyl-histidylhydrazide in patented formulations thereof, demonstrate the Schiff bases' transglycating activities concomitant with glycation site specific antioxidant activities and protection of proprietary antioxidant enzymes in the skin during aging and with diabetes lesions. During oral supplementation with stabilized from enzymatic hydrolysis carnosine (Can-C Plus® formulation), the skin parameters investigated showed a continuous and significant improvement in the active group during the 3 months of supplementation as compared to placebo. Visual investigation showed improvement of the overall skin appearance and a reduction of fine lines. No treatment-related side effects were reported. The finding that already-formed AGE cross-links can be pharmacologically severed and attendant pathology thereby reversed by non-hydrolized carnosine or carcinine in patented oral formulations thereof has broad implications for the skin beautification and therapeutics of the complications of diabetes and skin diseases associated with aging.

Carnosine and its possible roles in nutrition and health.

The dipeptide carnosine has been observed to exert antiaging activity at cellular and whole animal levels. This review discusses the possible mechanisms by which carnosine may exert antiaging action and considers whether the dipeptide could be beneficial to humans. Carnosine's possible biological activities include scavenger of reactive oxygen species (ROS) and reactive nitrogen species (RNS), chelator of zinc and copper ions, and antiglycating and anticross-linking activities. Carnosine's ability to react with deleterious aldehydes such as malondialdehyde, methylglyoxal, hydroxynonenal, and acetaldehyde may also contribute to its protective functions. Physiologically carnosine may help to suppress some secondary complications of diabetes, and the deleterious consequences of ischemic-reperfusion injury, most likely due to antioxidation and carbonyl-scavenging functions. Other, and much more speculative, possible functions of carnosine considered include transglutaminase inhibition, stimulation of proteolysis mediated via effects on proteasome activity or induction of protease and stress-protein gene expression, upregulation of corticosteroid synthesis, stimulation of protein repair, and effects on ADP-ribose metabolism associated with sirtuin and poly-ADP-ribose polymerase (PARP) activities. Evidence for carnosine's possible protective action against secondary diabetic complications, neurodegeneration, cancer, and other age-related pathologies is briefly discussed.

Biochemical properties of new synthetic carnosine analogues containing the residue of 2,3-diaminopropionic acid: the effect of N-acetylation.

Three novel carnosine analogues 7-9 containing the residue of L(+)2,3-diaminopropionic acid with different degree of N-acetylation instead of beta-alanine have been synthesized and characterized. Comparative analysis of hydrolysis by carnosinase revealed that the mono- and bis-acetylated compounds 8 and 9 are resistant to enzymatic hydrolysis and act as competitive inhibitors of this enzyme. The hydroxyl radical scavenging potential of the three analogues was evaluated by their ability to inhibit iron/H(2)O(2)-induced degradation of deoxyribose. The second-order rate constants of the reaction of compounds 7-9 with hydroxyl radical were almost identical to that of carnosine. These compounds were also found to act as protective agents against peroxynitrite-dependent damage as assessed by their ability to prevent nitration of free tyrosine induced by this species.

Antioxidative constituents of Rosmarinus officinalis and Salvia officinalis. II. Isolation of carnosic acid and formation of other phenolic diterpenes.

The phenolic diterpene carnosic acid appears to be the main substance for general oxidation leading to artifacts with gamma- or delta-lactone structure in extracts of Rosmarinus officinalis and Salvia officinalis. Until now it was only possible to prepare carnosic acid by hydrogenolysis of carnosol. A semipreparative HPLC method has been developed isolating carnosic acid among other phenolic diterpenes. The separated substances were identified by 13C-nuclear magnetic resonance (NMR), 1H-NMR, mass and IR spectroscopy. Conversion of carnosic acid and carnosol to other phenolic diterpenes was investigated by HPLC.