Assessment of Techno-Functional and Nutraceutical Potential of Tomato (Solanum lycopersicum) Seed Meal.

Tomato (Solanum lycopersicum) is a widely consumed fruit all around the world. The industrial exploitation of tomato generates a lot of waste. Most of the utilization of tomato seeds waste is focused on animal feeding, as well as a food ingredient aimed to increase the protein content, and raw material for some organic bioactive component extraction. The aim of this work was to evaluate the techno-functional properties of tomato seed meal (TSM) and its nutraceutical properties after applying defatting processing (TSMD), and to evaluate the nutraceutical properties after a fermentation processing (TSMDF) by Lactobacillus sp. The results showed that, at alkaline conditions (pH 8-9), the techno-functional properties for TSM and TSMD improved. In comparison with TSM, TSMD showed higher water holding capacity (WHC ≈32%), higher oil holding capacity (OHC ≈13%), higher protein solubility (49-58%), more than 10 times foaming activity (FA), more than 50 times foam stability (Fst), as well as an improved emulsifying activity (EA) and emulsion stability (Est) wich were better at pH 9. Regarding the nutraceutical properties, after 48 h of fermentation (TSMDF), the antioxidant activity was doubled and a significant increase in the iron chelating activity was also observed. During the same fermentation time, the highest angiotensin-converting enzyme inhibition (ACEI) was achieved (IC50 73.6 µg/mL), more than 10 times higher than TSMD, which leads to suggest that this fermented medium may be a powerful antihypertensive. Therefore, the strategy proposed in this study could be an option for the exploitation of tomato wastes.

Assessment of taste masking of captopril by ion-exchange resins using electronic gustatory system.

The objective of the study was to mask the unpleasant taste of captopril (CPT). Taste masking was achieved by complexation of CPT with a basic ion exchange resin, Dowex® 66, using the batch method. Dowex® 66 was used for the adsorption of CPT, and physical and chemical parameters of the CPT resinates complex were evaluated. A central composite design was used to generate the experiments for the manufacture of resinates using different process and formulation variables. In vitro dissolution studies were performed for 2 h in 0.01N HCl (pH 1.6) using USP Apparatus I. The compatibility of CPT and the resin was evaluated by Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD). The resinates were evaluated for micromeritic properties and further characterised using FTIR, DSC, and PXRD. Response surface methodology was used to determine the significance of input variables on the CPT content and release. The CPT resin ratio was found to have a significant impact on content of the resinates and on CPT release. The formulations were also studied for taste masking ability by means of an electronic gustatory system - electronic tongue.

Isolation and Characterization of Angiotensin Converting Enzyme Inhibitory Peptides from Peach Seed Hydrolysates: In Vivo Assessment of Antihypertensive Activity.

A peptide fraction with molecular masses below 3 kDa (PSH-3 kDa) from a peach seed hydrolysate demonstrated high angiotensin converting enzyme (ACE) inhibitory activity (concentration to inhibit 50% ACE (IC50) = 16.4 µg/mL) in our previous work. This work proposes a further study of this highly active fraction. RP-HPLC enabled two fractions (F3 and F4) with high inhibitory activity (IC50 = 2.0 ± 0.5 and 1.2 ± 0.2 µg/mL, respectively) to be isolated. Peptide analysis by LC-Q-TOF-MS/MS using reverse-phase and hydrophilic interaction chromatography enabled 33 peptides within both fractions to be identified. Among them, peptide isoleucine-tyrosine-serine-proline-histidine (IYSPH) showed the highest capacity. The lack of cytotoxicity of peptides was demonstrated in three different cell lines (HeLa, HT-29, and HK-2). Oral administration of PSH-3 kDa fraction or peptide IYSPH caused a significant systolic blood pressure reduction (-30 mmHg) on spontaneously hypertensive rats after 3-6 h treatment.

"Ideal correlations" for biological activity of peptides.

Sequences of one-symbol abbreviations of amino acids are applied as the basis to build up predictive model of Angiotensin converting enzyme (ACE) inhibitory activity of dipeptides and antibacterial activity of group of polypeptides. The developed models are one-variable correlations between biological activity and descriptors calculated with so-called correlation weights of amino acids. The numerical data on the correlation weights are obtained by the Monte Carlo method. The Index of Ideality of Correlation (IIC) is a mathematical function of (i) the determination coefficient; and (ii) sums of positive and negative values of "observed minus predicted" endpoints values. The obtained results confirm that IIC can be applied to improve predictive potential of models for ACE inhibitor activity of dipeptides and antibacterial activity of polypeptides.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening.

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.

Improving efficiency of an angiotensin converting enzyme inhibitory peptide as multifunctional peptides.

Bioactive peptides have been defined as specific protein fragments that have numerous biological activities. The aim of this study was to introduce three multifunctional peptides. Hence, we used rabbit lung angiotensin converting enzyme (ACE) inhibitor peptide AFKDEDTEEVPFR to prepare two analogous peptides KDEDTEEVP and KDEDTEEVH. ACE inhibitory, antioxidant, and antimicrobial activities of three synthetic peptides were investigated. Among the three peptides, KDEDTEEVP exhibited the highest ACE inhibitory activity with IC50 value of 69.63 ± 2.51 µM. Furthermore, the results of fluorescence spectroscopy and molecular modeling showed that KDEDTEEVP had more affinity to ACE than other peptides. The peptide of KDEDTEEVH showed the strongest antioxidant scavenging capacity on DPPH radicals (EC50 = 135 ± 9.62 µM), hydroxyl radicals (EC50 = 144 ± 8.73 µM), and ABTS radicals (EC50 = 62 ± 4.52%). Moreover, it showed the highest activity in iron-chelating test (EC50 = 226 ± 14.13 µM) and could also effectively inhibit the peroxidation of linoleic acid. The antimicrobial activity results showed that KDEDTEEVH had higher efficiency against Gram-positive than Gram-negative bacteria with MIC values of higher than 205 ± 10.75 µM. Although there was not a direct correlation between ACE inhibitor and antioxidant activity for analogous peptides, both analogous peptides exhibited more efficiency than the mother peptide. Thus, they can be considered as multifunctional peptides and would be beneficial ingredient to be used in food and drug industry.

Evaluation of nutritional value, characteristics, functional properties of Cymodocea nodosa and its benefits on health diseases.

BACKGROUND: Nutritional fact study has prime importance to make the species edible and commercially viable to the food consumers. This is the first report that indicates the chemical characterization, functional, antioxidant and antihypertensive properties of Cymodocea nodosa to evaluate its nutritional status. METHODS: Physico-chemical determination was determined by colorimetric and spectroscopic analysis. The functional and texture properties were evaluated since a desirable texture should be retained. Bioactive substances were determined by liquid chromatography-high resolution electrospray ionization mass spectrometry HPLC-DAD-ESI/MS2 analysis. Health benefit of this plant was highlighting by the antioxidant and antihypertensive potentials. RESULTS: Results showed that the seagrass powder was characterized by a high content of fibers (56.4%), the fatty acids profile was dominated by the oleic acid, which represents about 62.0% of the total fatty acids and the functional properties proved important values of swelling capacity (6.71 ± 0.2) and water holding capacity (12.26 ± 0.25), that were comparable to those of some foodstuffs. Finally, the physico-chemical analysis shows the wealth in phenolic compounds, that could be explained by the high antioxidant and antihypertensive ability which was concentration dependent. CONCLUSION: The results from this study suggested that this marine plant could be utilized as a healthy food item for human consumption.

A series of N-of-1 trials to assess the therapeutic interchangeability of two enalapril formulations in the treatment of hypertension in Addis Ababa, Ethiopia: study protocol for a randomized controlled trial.

BACKGROUND: Hypertension is one of the leading causes of morbidity and mortality in Ethiopia. Treatment usually involves lifelong medication use. Enalapril is a common drug for the treatment of hypertension in Ethiopia. However, the drug is expensive and, therefore, there is limited capacity for people to afford the treatment. Locally produced Enalapril is a cost-effective solution to treat the disease. However, as local medicines regulation does not include bioequivalence tests on locally produced drugs, physicians and patients need assurance about the effectiveness and safety of local generics. Evidence on therapeutic equivalence is needed on these untested local drugs. METHODS: This is a hospital-based, randomized, partially blinded, three-cycle crossover trial in single patients, comparing a locally produced version of enalapril with enalapril imported from Europe. Patients involved in this trial are not blinded, as there is no local facility to produce relatively small numbers of placebos or encapsulated drugs. To ensure blinding of study investigators and data analysts, study medications are prepared by an independent pharmacy unit using opaque medication packaging. The importance of maintaining blinding is also part of patient pre-trial education. Each N-of-1 trial will consist of three successive 14-day treatment pairs, each pair comprising 7 days of 5-20 mg local and 7 days of 5-20 mg imported enalapril taken once daily in the morning. The primary outcome will be the average difference in systolic blood pressure as measured by home blood pressure measurements. DISCUSSION: The number of locally produced products, such as enalapril, being approved without proof of bioequivalence is dramatically increasing. By bridging the information gap on bioequivalence, the trial will give rigorous evidence on therapeutic equivalence of locally produced enalapril in the treatment of hypertension. If there is no difference, the hypothesized result, then patients can take the local medicine with confidence. This trial will also will determine whether aggregated N-of-1 studies are feasible to evaluate untested generic drugs in resource-limited countries where bioequivalence testing centers are unavailable. TRIAL REGISTRATION NUMBER: Australian and New Zealand Clinical Trial Registry, ID: ACTRN12616001088437p . Registered on 12 August 2016.

Extraction and Quantification of Sinapinic Acid from Irish Rapeseed Meal and Assessment of Angiotensin-I Converting Enzyme (ACE-I) Inhibitory Activity.

Phenolic compounds, including phenolic acids, are known to play a protective role against the development of cardiovascular disease. The aim of this work was to generate a phenolic acid extract from Irish rapeseed meal, to determine the quantity of sinapinic acid (SA) in this fraction and to assess the ability of this fraction to inhibit the enzyme angiotensin-I converting enzyme (ACE-I; EC 3.4.15.1). A crude phenolic extract (fraction 1), free phenolic acid containing extract (fraction 2), and an extract containing phenolic acids liberated from esters (fraction 3) were generated from Irish rapeseed meal using a methanol:acetone:water solvent mixture (7:7:6). The total phenolic content (TPC) of each extract was determined and proximate analysis performed to determine the fat, moisture, and protein content of these extracts. Nuclear magnetic resonance (1H NMR) spectroscopy was used to quantify the level of SA in extract 3, which inhibited ACE-I by 91% ± 0.08 when assayed at a concentration of 1 mg/mL, compared to the control, captopril, which inhibited ACE by 97% ± 0.01 when assayed at a concentration of 1 mg/mL.

Sulfated polysaccharides from common smooth hound: Extraction and assessment of anti-ACE, antioxidant and antibacterial activities.

The present study investigates biological activities of sulfated polysaccharides (SPs) isolated from smooth hound by precipitation with cetylpyridinium chloride (SP1) or ethanol (SP2). SP1 showed the highest amounts of sulfated groups (10.2%) and proteins (7.84%) and high molecular weight sugars. Infrared spectroscopic analysis showed typical peaks of sulfated polysaccharides, particularly for the SP1 that was characterized by the presence of O=S=O groups and acetyl groups. Interestingly, SPs displayed important angiotensin I converting enzyme (ACE) inhibitory (IC50=1.04 and 0.75mg/ml for SP1 and SP2, respectively), antibacterial (Gram+ and Gram-) and antioxidant activities (reducing power, metal chelating activity, ß-carotene bleaching inhibition and DNA nicking assay). Moreover, SPs fractionation by DEAE-cellulose column chromatography showed one peak during the buffer elution phase and three major fractions during the linear gradient of NaCl. The overall data suggested that SPs could be used as natural antioxidant, antimicrobial and anti-ACE ingredient to formulate functional foods.

Purification and identification of angiotensin I-converting enzyme-inhibitory peptides from apalbumin 2 during simulated gastrointestinal digestion.

BACKGROUND: Bee larvae are considered to be an important reservoir for proteins. However, little attention has been paid to the release of potential bioactive peptides from bee larva proteins. In this study the major protein in bee larvae was hydrolyzed in vitro by gastrointestinal enzymes. The peptide profile of the hydrolysis was characterized by gel filtration chromatography and tricine-SDS-PAGE. Furthermore, the bioactive peptide was isolated and identified by Q-TOF-MS/MS. RESULTS: The major bee larva protein was identified as apalbumin 2 and was more digestible into peptides with molecular weights lower than 3 kDa. The hydrolysate obtained after 3 h of digestion exhibited angiotensin I-converting enzyme (ACE)-inhibitory activity and was purified sequentially by gel filtration and RP-HPLC. The molecular weights of peptide fractions with ACE-inhibitory activity were distributed between 0.5 and 1.5 kDa. A novel peptide with highest ACE-inhibitory activity (IC50 54.9 µmol L(-1) ) was purified by further RP-HPLC. The amino acid sequence of this peptide was identified as LLKPY (632.40 Da). CONCLUSION: ACE-inhibitory peptides could be formed from bee larvae through gastrointestinal digestion. The most active peptide (LLKPY) is potentially useful as a therapeutic agent in treating hypertension.

New ketomethylene inhibitor analogues: synthesis and assessment of structural determinants for N-domain selective inhibition of angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase containing two homologous domains. While the C-domain plays a major role in blood pressure regulation, the N-domain hydrolyzes the antifibrotic agent N-acetyl-Ser-Asp-Lys-Pro. Thus, N-domain selective (N-selective) inhibitors could be useful in the treatment of conditions relating to excessive tissue fibrosis. New keto-ACE analogues were designed that contained functionalities considered important for N-selective inhibitor RXP407 binding, namely, a P(2) Asp, N-acetyl group, and C-terminal amide. Such functionalities were incorporated to assess the structural determinants for N-selective binding in a novel inhibitor template. Inhibitors containing a C-terminal amide and modified P(2)' group were poor inhibitors of the N-domain, with several of these displaying improved inhibition of the C-domain. Molecules with both a C-terminal amide and P(2) Asp were also poor inhibitors and not N-selective. Compounds containing a free C-terminus, a P(2) Asp and protecting group displayed a change of more than 1000-fold N-selectivity compared with the parent molecule. Molecular docking models revealed interaction of these P(2) groups with S(2) residues Tyr369 and Arg381. This study emphasizes the importance of P(2) functionalities in allowing for improved N-selective binding and provides further rationale for the design of N-selective inhibitors, which could be useful in treating tissue fibrosis.

Assessment of liquid captopril formulations used in children.

OBJECTIVE: Unlicensed liquid captopril formulations are commonly used to treat children with heart disease. This study assessed the bioequivalence of two liquid preparations against a licensed tablet form. DESIGN: An open label, single dose, three-treatment, three-period, crossover trial. SETTING: Outpatient. PATIENTS: Healthy adult volunteers (n=18). INTERVENTIONS: Each subject was randomly assigned to one of six dosing sequences, and dosed with 25 mg captopril on each of three dosing visits separated by a washout of at least 14 days. Blood samples for pharmacokinetic analysis were taken at regular intervals (0 min to 10 h) post-dose. MAIN OUTCOME MEASURES: Bioequivalence of the formulations would be concluded if the 90% CI for the estimated ratio of the means of C(max) (maximum plasma concentrations) and area under curve(AUC) (extent of absorption) lay entirely within the range 0.8 to 1.25 RESULTS: Both liquid formulations failed the bioequivalence assessment with respect to C(max) and AUC. The 90% CI of the mean ratios of liquid/licensed tablet for both C(max) and AUC, fell outside the 0.8 to 1.25 limits. There was also considerable within-subject variability in C(max) (97.5%) and AUC (78.5%). CONCLUSIONS: Unlicensed captopril formulations are not bioequivalent to the licensed tablet form, or to each other, and so cannot be assumed to behave similarly in therapeutic use. Thus formulation substitution must be done with care and may require a period of increased monitoring of the patient. There is also significant within-subject variability in performance which has clinical implications with respect to titrating to an optimum therapeutic dose.

Development and bioavailability assessment of ramipril nanoemulsion formulation.

The objective of our investigation was to design a thermodynamically stable and dilutable nanoemulsion formulation of Ramipril, with minimum surfactant concentration that could improve its solubility, stability and oral bioavailability. Formulations were taken from the o/w nanoemulsion region of phase diagrams, which were subjected to thermodynamic stability and dispersibility tests. The composition of optimized formulation was Sefsol 218 (20% w/w), Tween 80 (18% w/w), Carbitol (18% w/w) and standard buffer solution pH 5 (44% w/w) as oil, surfactant, cosurfactant and aqueous phase, respectively, containing 5 mg of ramipril showing drug release (95%), droplet size (80.9 nm), polydispersity (0.271), viscosity (10.68 cP), and infinite dilution capability. In vitro drug release of the nanoemulsion formulations was highly significant (p<0.01) as compared to marketed capsule formulation and drug suspension. The relative bioavailability of ramipril nanoemulsion to that of conventional capsule form was found to be 229.62% whereas to that of drug suspension was 539.49%. The present study revealed that ramipril nanoemulsion could be used as a liquid formulation for pediatric and geriatric patients and can be formulated as self-nanoemulsifying drug delivery system (SNEDDS) as a unit dosage form.

High-throughput quantification of perindopril in human plasma by liquid chromatography/tandem mass spectrometry: application to a bioequivalence study.

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of perindopril in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 369/172 for perindopril and m/z 417/234 for the internal standard. The method exhibited a linear dynamic range of 0.1-100 ng/mL for perindopril in human plasma. The lower limit of quantification was 0.1 ng/mL with a relative standard deviation of less than 6.1%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 450 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.

Symmetry considerations in Markovian chemicals 'in silico' design (MARCH-INSIDE) I: central chirality codification, classification of ACE inhibitors and prediction of sigma-receptor antagonist activities.

The MARCH-INSIDE methodology has been generalized, by means of an exponential central symmetry factor, to codify chemical structure information for chiral drugs. In order to test the potential of this novel approach in drug design we have modeled the angiotensin-converting enzyme inhibitory activity of perindoprilate's sigma-stereoisomer combinatorial library. A linear discriminant analysis (LDA) model classifies correctly 83.33% of active compounds and 94.12% of non-active ones in a training set, results that represent a total of 91.3% accuracy in classification. On the other hand, the model classifies 83.33% of these compounds in the predicting series. Only three isomers (those with higher activity) were used in the predicting set and the model classified all three very well. Similar predictive behavior was observed in a leave-1-out cross validation experiment. Canonical regression analysis corroborated the statistical quality of the models (Rcanc=0.79, with a P-level<0.000) and was also used to compute biological activity canonical scores for each compound. Finally, prediction of the biological activities of chiral 3-(3-hydroxyphenyl)piperidines, which are sigma-receptor antagonists, by linear regression analysis was carried out. The model was statistically significant (R=0.963, S=0.29, P<0.00) and can be considered as a preliminary comparative study between MARCH-INSIDE and Chiral Topologic descriptors. Application of the Student test permits the detection of non-symmetric properties within the data set and justified the requirement of non-symmetric (for pairs of enantiomers) molecular descriptors. The MARCH-INSIDE model showed very good stability to data variation in the leave-1-out cross validation experiment (Scv=0.32).

QXP: powerful, rapid computer algorithms for structure-based drug design.

New methods for docking, template fitting and building pseudo-receptors are described. Full conformational searches are carried out for flexible cyclic and acyclic molecules. QXP (quick explore) search algorithms are derived from the method of Monte Carlo perturbation with energy minimization in Cartesian space. An additional fast search step is introduced between the initial perturbation and energy minimization. The fast search produces approximate low-energy structures, which are likely to minimize to a low energy. For template fitting, QXP uses a superposition force field which automatically assigns short-range attractive forces to similar atoms in different molecules. The docking algorithms were evaluated using X-ray data for 12 protein-ligand complexes. The ligands had up to 24 rotatable bonds and ranged from highly polar to mostly nonpolar. Docking searches of the randomly disordered ligands gave rms differences between the lowest energy docked structure and the energy-minimized X-ray structure, of less than 0.76 A for 10 of the ligands. For all the ligands, the rms difference between the energy-minimized X-ray structure and the closest docked structure was less than 0.4 A, when parts of one of the molecules which are in the solvent were excluded from the rms calculation. Template fitting was tested using four ACE inhibitors. Three ACE templates have been previously published. A single run using QXP generated a series of templates which contained examples of each of the three. A pseudo-receptor, complementary to an ACE template, was built out of small molecules, such as pyrrole, cyclopentanone and propane. When individually energy minimized in the pseudo-receptor, each of the four ACE inhibitors moved with an rms of less than 0.25 A. After random perturbation, the inhibitors were docked into the pseudo-receptor. Each lowest energy docked structure matched the energy-minimized geometry with an rms of less than 0.08 A. Thus, the pseudo-receptor shows steric and chemical complementarity to all four molecules. The QXP program is reliable, easy to use and sufficiently rapid for routine application in structure-based drug design.