Ramipril Improves Endothelial Function and Increases the Number of Endothelial Progenitor Cells in Patients With Systemic Lupus Erythematosus.

BACKGROUND/OBJECTIVES: Endothelial dysfunction and reduced number of endothelial progenitor cells (EPCs) in peripheral blood are contributing factors to cardiovascular disease in systemic lupus erythematosus (SLE) patients. Endothelial progenitor cell proliferation is regulated by vascular endothelial growth factor (VEGF). Angiotensin-converting enzyme inhibitors reduce cardiovascular mortality in patients with coronary heart disease. METHODS: This was a randomized trial including 37 female SLE patients without cardiovascular risk factors allocated into 2 groups: 19 patients received ramipril 10 mg/d for 12 weeks (IG) and 18 patients maintained without ramipril (CG). Endothelial function was assessed by brachial artery ultrasound measuring flow-mediated dilation, and EPCs were quantified by flow cytometry and cell culture, at baseline and after 12 weeks. Serum VEGF levels were measured by enzyme-linked immunosorbent assay. Statistical analysis was intention to treat. p < 0.05 was considered significant. RESULTS: After 12 weeks, higher flow-mediated dilation (6.17% vs. 11.14%, p < 0.001) was observed in IG, without change in CG (5.37% vs. 5.02%, p = 0.630). Higher number of EPC colony-forming units was also observed in IG (21.3 ± 10.4 vs. 31.6 ± 8.5, p < 0.001), without difference in CG ( p = 0.714). No difference was found in EPCs evaluated by flow cytometry. Vascular endothelial growth factor level increased after 12 weeks in IG ( p = 0.048), with no difference in CG ( p = 0.661). CONCLUSION: Ramipril improved endothelial function and increased the numbers of EPCs evaluated by cell culture and VEGF levels in SLE patients without cardiovascular risk factors. These data suggest that angiotensin-converting enzyme inhibitor bring an extra benefit beyond the hypotensive action and should be considered as a preferred antihypertensive drug in SLE patients.

ACE2, the Receptor that Enables Infection by SARS-CoV-2: Biochemistry, Structure, Allostery and Evaluation of the Potential Development of ACE2 Modulators.

Angiotensin converting enzyme 2 (ACE2) is the human receptor that interacts with the spike protein of coronaviruses, including the one that produced the 2020 coronavirus pandemic (COVID-19). Thus, ACE2 is a potential target for drugs that disrupt the interaction of human cells with SARS-CoV-2 to abolish infection. There is also interest in drugs that inhibit or activate ACE2, that is, for cardiovascular disorders or colitis. Compounds binding at alternative sites could allosterically affect the interaction with the spike protein. Herein, we review biochemical, chemical biology, and structural information on ACE2, including the recent cryoEM structures of full-length ACE2. We conclude that ACE2 is very dynamic and that allosteric drugs could be developed to target ACE2. At the time of the 2020 pandemic, we suggest that available ACE2 inhibitors or activators in advanced development should be tested for their ability to allosterically displace the interaction between ACE2 and the spike protein.

Biochemical Properties of a Partially Purified Protease from Bacillus sp. CL18 and Its Use to Obtain Bioactive Soy Protein Hydrolysates.

Microbial proteases are relevant biocatalysts with diverse applications. Production of protein hydrolysates is recently focused, since they might display biological activities. Therefore, the extracellular protease from Bacillus sp. CL18 was partially purified through ammonium sulfate precipitation (25-50% saturation) and gel filtration chromatography, with a 60.7-fold purification (40,593 U/mg protein) and 21.3% recovery. The partially purified protease (PPP) was characterized as a serine protease, with optimal activity at 51-59 °C and pH 7.4-8.8 and low thermal stability. Thermal inactivation followed first-order kinetics. PPP depended on Ca2+ for higher thermal stability, depicted by increases in half-lives (t1/2), activation energy (Ea), and free energy (ΔG#) for kinetic inactivation. PPP preferentially hydrolyzed casein > soy protein isolate (SPI) >>> keratinous materials. SPI hydrolysis by PPP was further investigated, and the obtained hydrolysates exhibited increased in vitro bioactivities. Hydrolysates displayed antioxidant capacities through the scavenging of synthetic organic radicals and Fe3+-reducing ability. In addition, hydrolysates inhibited the activities of dipeptidyl peptidase IV (DPP IV) and angiotensin-converting enzyme (ACE), suggesting antidiabetic and antihypertensive potentials, respectively. From its biochemical properties, PPP might be used to produce protein hydrolysates with multifunctional bioactivities. Both PPP and SPI hydrolysates can find applications in food biotechnology.

Transforming Non-Selective Angiotensin-Converting Enzyme Inhibitors in C- and N-domain Selective Inhibitors by Using Computational Tools.

The two-domain dipeptidylcarboxypeptidase Angiotensin-I-converting enzyme (EC 3.4.15.1; ACE) plays an important physiological role in blood pressure regulation via the reninangiotensin and kallikrein-kinin systems by converting angiotensin I to the potent vasoconstrictor angiotensin II, and by cleaving a number of other substrates including the vasodilator bradykinin and the anti-inflammatory peptide N-acetyl-SDKP. Therefore, the design of ACE inhibitors is within the priorities of modern medical sciences for treating hypertension, heart failures, myocardial infarction, and other related diseases. Despite the success of ACE inhibitors for the treatment of hypertension and congestive heart failure, they have some adverse effects, which could be attenuated by selective domain inhibition. Crystal structures of both ACE domains (nACE and cACE) reported over the last decades could facilitate the rational drug design of selective inhibitors. In this review, we refer to the history of the discovery of ACE inhibitors, which has been strongly related to the development of molecular modeling methods. We stated that the design of novel selective ACE inhibitors is a challenge for current researchers which requires a thorough understanding of the structure of both ACE domains and the help of molecular modeling methodologies. Finally, we performed a theoretical design of potential selective derivatives of trandolaprilat, a drug approved to treat critical conditions of hypertension, to illustrate how to use molecular modeling methods such as de novo design, docking, Molecular Dynamics (MD) simulations, and free energy calculations for creating novel potential drugs with specific interactions inside nACE and cACE binding sites.

Fluorofenidone inhibits apoptosis of renal tubular epithelial cells in rats with renal interstitial fibrosis.

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.

Influence of a Co-culture of Streptococcus thermophilus and Lactobacillus casei on the Proteolysis and ACE-Inhibitory Activity of a Beverage Based on Reconstituted Goat Whey Powder.

The potential for reusability of whey is of concern due to its substantial nutritional value and, second, in view of the need to reduce environmental impact. The use of goat whey powder in the production of a fermented dairy beverage using a Lactobacillus casei culture as adjunct shows a good prospect of a food product with hypotensive activity. This study investigated the microbial viability, proteolysis and angiotensin-converting enzyme (ACE) inhibitory activity of a fermented dairy beverage produced with goat whey powder and a probiotic culture of L. casei BGP93 co-cultured with Streptococcus thermophilus TA-40. The probiotic beverage exhibited no significant difference from the control (absence of L. casei) with regard to titratable acidity, S. thermophilus viability and proteolysis degree during 21 days of storage (P > 0.05). During this period, the beverage maintained L. casei at appropriate levels (> 7 log cfu ml-1), thereby qualifying as a potential probiotic product. Although both control and probiotic beverages exhibited ACE inhibitory activity, as a result of proteolysis of whey proteins during fermentation, significant increased ACE inhibitory activity was found for the beverage with added probiotic L. casei (P < 0.05). The probiotic beverage has potency as a functional food candidate to be included in a dietary strategy aiming at prevention and control of hypertension.

Fluorofenidone inhibits apoptosis of renal tubular epithelial cells in rats with renal interstitial fibrosis

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.

A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain--Potential BPP-like precursor protein?

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.

Hydrolysates of sheep cheese whey as a source of bioactive peptides with antioxidant and angiotensin-converting enzyme inhibitory activities.

Enzymatic proteolysis may be employed to release bioactive peptides, which have been investigated for potential benefits from both technological and human health perspectives. In this study, sheep cheese whey (SCW) was hydrolyzed with a protease preparation from Bacillus sp. P7, and the hydrolysates were evaluated for antioxidant and angiotensin I-converting enzyme (ACE)-inhibitory activities. Soluble protein and free amino acids increased during hydrolysis of SCW for up to 4h. Antioxidant activity of hydrolysates, evaluated by the 2,2'azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid radical scavenging method, increased 3.2-fold from 0 h (15.9%) to 6h of hydrolysis (51.3%). Maximum Fe(2+) chelation was reached in 3h hydrolysates, and the reducing power peaked at 1h of hydrolysis, representing 6.2 and 2.1-fold increase, respectively, when compared to that of non-hydrolyzed SCW. ACE inhibition by SCW (12%) was improved through hydrolysis, reaching maximal values (55% inhibition) in 4h, although 42% inhibition was already observed after 1h hydrolysis. The peptide LAFNPTQLEGQCHV, derived from ß-lactoglobulin, was identified from 4-h hydrolysates. Such a biotechnological approach might be an interesting strategy for SCW processing, potentially contributing to the management and valorization of this abundant dairy byproduct.

Production of feather hydrolysates with antioxidant, angiotensin-I converting enzyme- and dipeptidyl peptidase-IV-inhibitory activities.

The antioxidant and antihypertensive activities of feather hydrolysates obtained with the bacterium Chryseobacterium sp. kr6 were investigated. Keratin hydrolysates were produced with different concentrations of thermally denatured feathers (10-75 g l(-1)) and initial pH values (6.0-9.0). Soluble proteins accumulated in high amounts in media with 50 and 75 g l(-1) of feathers, reaching values of 18.5 and 22 mg ml(-1), respectively, after 48 hours of cultivation. In media with 50 g l(-1) of feathers, initial pH had minimal effect after 48 hours. Maximal protease production was observed after 24 hours of cultivation, and feather concentration and initial pH values showed no significant effect on enzyme yields at this time. Feather hydrolysates displayed in vitro antioxidant properties, and optimal antioxidant activities were observed in cultures with 50 g l(-1) feathers, at initial pH 8.0, after 48 hours growth at 30°C. Also, feather hydrolysates were demonstrated to inhibit the angiotesin I-converting enzyme by 65% and dipeptidyl peptidase-IV by 44%. The bioconversion of an abundant agroindustrial waste such as chicken feathers can be utilized as a strategy to obtain hydrolysates with antioxidant and antihypertensive activities. Feather hydrolysates might be employed as supplements in animal feed, and also as a potential source of bioactive molecules for feed, food and drug development.

Relationship between renal injury and the antagonistic roles of angiotensin-converting enzyme (ACE) and ACE2.

Angiotensin-converting enzyme 2 (ACE2), a newly discovered carboxypeptidase in the renin-angiotensin system (RAS), antagonizes ACE activity and plays an active role during tissue injury. Yet the mechanism of its action is not well known. Using a streptozotocin (STZ)-induced renal injury rat model, we investigated the relationship between renal injury and the antagonism between ACE and ACE2. We assayed the levels of urea nitrogen, urine glucose, creatinine, and protein, Ace2, Ace, angiotensin II type 1 receptor (At1) and Mas receptor mRNA, and renal and plasma angiotensin II (Ang II) in STZ-treated and untreated rats. We also used histology and immunohistochemistry to assess glomerular injury and ACE2 glomerular and cortical expression. The amounts of urea nitrogen, urine glucose, creatinine, and protein were significantly higher in STZ-treated rats than in control rats (P < 0.01). There were significant pathological changes in the kidney upon STZ-treatment. Ace2 and Ace mRNA levels were significantly higher in STZ-treated rats than in control rats (P < 0.05 and P = 0.05, respectively). There was no significant difference in the Mas receptor and At1 mRNA levels in the 2 groups, although At1 levels showed an increase upon STZ-treatment. The Ang II level in the renal cortical tissue and plasma of STZ-treated rats was higher than that of control rats (P < 0.05). The increase in Ace mRNA levels was higher than that of Ace2 mRNA levels, leading to an elevated Ace/Ace2 ratio. Together, these data suggest that the ACE-Ang II-AT1 axis is the dominant axis in severe kidney injury.

Proline rich-oligopeptides: diverse mechanisms for antihypertensive action.

Bradykinin-potentiating peptides from Bothrops jararaca (Bj) discovered in the early 1960s, were the first natural inhibitors of the angiotensin-converting enzyme (ACE). These peptides belong to a large family of snake venom proline-rich oligopeptides (PROs). One of these peptides, Bj-PRO-9a, was essential for defining ACE as effective drug target and development of captopril, an active site-directed inhibitor of ACE used worldwide for the treatment of human arterial hypertension. Recent experimental evidences demonstrated that cardiovascular effects exerted by different Bj-PROs are due to distinct mechanisms besides of ACE inhibition. In the present work, we have investigated the cardiovascular actions of four Bj-PROs, namely Bj-PRO-9a, -11e, -12b and -13a. Bj-PRO-9a acts upon ACE and BK activities to promote blood pressure reduction. Although the others Bj-PROs are also able to inhibit the ACE activity and to potentiate the BK effects, our results indicate that antihypertensive effect evoked by them involve new mechanisms. Bj-PRO-11e and Bj-PRO-12b involves induction of [Ca(2+)]i transients by so far unknown receptor proteins. Moreover, we have suggested argininosuccinate synthetase and M3 muscarinic receptor as targets for cardiovascular effects elicited by Bj-PRO-13a. In summary, the herein reported results provide evidence that Bj-PRO-mediated effects are not restricted to ACE inhibition or potentiation of BK-induced effects and suggest different actions for each peptide for promoting arterial pressure reduction. The present study reveals the complexity of the effects exerted by Bj-PROs for cardiovascular control, opening avenues for the better understanding of blood pressure regulation and for the development of novel therapeutic approaches.

Peptides with angiotensin I converting enzyme (ACE) inhibitory activity generated from porcine skeletal muscle proteins by the action of meat-borne Lactobacillus.

Angiotensin I converting enzyme (ACE) inhibitory activity of peptides derived from the hydrolysis of sarcoplasmic and myofibrillar porcine proteins by the action of Lactobacillus sakei CRL1862 and Lactobacillus curvatus CRL705 (whole cells+cell free extracts) was investigated at 30°C for 36 h. The protein hydrolysates were subjected to RP-HPLC in order to fractionate the extracts for further evaluation of ACE inhibitory activity. Bioactive fractions were only found from the hydrolysis of sarcoplasmic proteins by both assayed lactobacilli strains. Identification of peptides contained in the bioactive fractions was carried out by tandem mass spectrometry using a nanoLC-ESI-QTOF instrument and the mascot search engine. From the four most active fractions obtained, a total of eighteen and fifty peptides were characterized from L. sakei CRL1862 and L. curvatus CRL705 protein hydrolysates, respectively. The sequence FISNHAY was generated by the proteolytic activity of the two lactobacilli species. Sequence similarity analyses between the peptides identified in this study and those previously identified as ACE inhibitory peptides and detailed in the BIOPEP database were outlined. Results suggest that meat-borne Lactobacillus were able to generate peptides with ACE inhibitory activity, highlighting their potential to be used in the development of functional fermented products. BIOLOGICAL SIGNIFICANCE: The results of this study would enable the obtention of porcine functional foods by applying lactic acid bacteria generating bioactive peptides. ACE inhibitory peptides obtained by the hydrolytic action of L. curvatus CRL705 and L. sakei CRL1862 on sarcoplasmic proteins were analyzed. Among them, the peptide FISNHAY exhibited the highest activity and its sequence has not yet been reported.

Simulated digestion of proanthocyanidins in grape skin and seed extracts and the effects of digestion on the angiotensin I-converting enzyme (ACE) inhibitory activity.

This study investigated the effect of in vitro gastrointestinal digestion on the stability and composition of flavan-3-ols from red grape skin and seed extracts (raw and purified, which are high in proanthocyanidins (PAs)). In addition, the effects of digestion on the angiotensin I-converting enzyme (ACE) inhibitory activities of these extracts were evaluated. The extracts were digested with a mixture of pepsin-HCl for 2 h, followed by a 2 h incubation with pancreatin and bile salts including a cellulose dialysis tubing (molecular weight cut-off 12 kDa) at 37°C with shaking in the dark and under N2. Under gastric conditions, the mean degree of polymerisation (mDP) of seed extracts, raw (mDP≈6, p<0.05), and purified (mDP≈10, p<0.05) was stable. The mDP of the raw skin extracts increased from 19 to 25 towards the end of the digestion. The PAs were significantly degraded (up to 80%) during the pancreatic digestion, yielding low-molecular-weight compounds that diffused into the serum-available fraction (mDP≈2). The overall mass transfer coefficient (K) of the seed extracts was 10(-7) m(2)/s. After simulated gastrointestinal digestion, over 80% of ACE inhibition by raw seed and skin extracts was preserved. However, the purified seed and skin extracts lost their ability to inhibit ACE after intestinal digestion.

Inhibición de la Enzima Convertidora de Angiotensina I con hidrolizados proteicos de Jatropha curcas / Angiotensin I-Converting Enzyme inhibition with protein hydrolysates from Jatropha curcas / Inibição da Enzima Conversora de Angiotensina I com hidrolisados proteicos de Jatropha curcas

Se evaluó la actividad inhibitoria in vitro de los hidrolizados proteínicos obtenidos a partir de la harina desgrasada y del aislado proteínico provenientes del grano de Jatropha curcas L. sobre la actividad de la enzima convertidora de angiotensina I (ECA-1), con la finalidad de emplearlos en un futuro para la formulación de alimentos funcionales. Los hidrolizados fueron obtenidos empleando alcalasa y el sistema enzimático pepsina-pancreatina. Se calculó la concentración media inhibitoria (IC50) para medir el grado de inhibición de la actividad enzimática de ECA-1. Fueron seleccionados los hidrolizados con el menor tiempo de hidrólisis (60 min) para evaluar la bioactividad, dado que las cinéticas de hidrólisis enzimática de la harina desgrasada y del aislado proteínico no encontraron diferencias significativas en el grado de hidrólisis para los tiempos de reacción en cada sistema (60, 90 y 120 min). Los valores de IC50 que presentaron el mejor efecto de inhibición sobre la ECA-I fueron 2,8 y 7,0 µg/mL, obtenidos a partir del aislado proteínico con la enzima alcalasa y con el sistema secuencial pepsina-pancreatina, respectivamente. Los hidrolizados de J. curcas podrían ser incorporados en la elaboración de alimentos funcionales y ser aplicados en tratamientos para personas con hipertensión por su efecto inhibitorio sobre la ECA-I.

In vitro angiotensin I-converting enzyme (ACE) inhibitory activity was evaluated in protein hydrolysates from defatted meal and protein isolate from Jatropha curcas L. Seed, in order to determine their potential inclusion in functional food formulation. Hydrolysates were produced using Alcalase® or a sequential pepsin-pancreatin enzymatic system. Mean inhibitory concentration (IC50) was used to measure the degree of ACE enzymatic activity inhibition. Bioactivity was evaluated in the hydrolysates with the lowest hydrolysis time (60 min) given that no differences in degree of hydrolysis in terms of reaction time in each system were observed (60, 90 and 120 min) in the enzymatic hydrolysis kinetics for the defatted meal and protein isolate. The protein isolate exhibited the highest inhibitory effect, as seen in the IC50 values: 2.8 µg/mL in the alcalase system and 7.0 µg/mL in the pepsin-pancreatin system. Hydrolysates from J. curcas seed exhibit ACE inhibition and could be incorporated into functional foods or treatments for those suffering hypertension.

Foi avaliada a atividade inibitória in vitro dos hidrolisados proteicos obtidos a partir da farinha desengordurada e do isolado proteico provenientes do grão de Jatropha curcas L. sobre a atividade da enzima conversora de angiotensina I (ECA-1), com o objetivo de utilizá-los num futuro para a formulação de alimentos funcionais. Os hidrolisados foram obtidos usando alcalase e o sistema enzimático pepsina-pancreatina. Foi calculada a concentração média inibitória (IC50) para medir o grau de inibição da atividade enzimática da ECA-1. Foram selecionados os hidrolisados com o menor tempo de hidrólise (60 min.) para avaliar a bioatividade visto que as cinéticas de hidrólise enzimática da farinha desengordurada e do isolado proteico não encontraram diferenças significativas no grau de hidrólise para os tempos de reação para cada sistema (60, 90 e 120 min.). Os valores de IC50 que apresentaram o melhor efeito de inibição sobre a ECA-I foram 2.8 e 7.0 µg/mL obtidos a partir do isolado proteico com a enzima alcalase e com o sistema sequencial pepsina-pancreatina respectivamente. Os hidrolisados de J. curcas poderiam ser incorporados na elaboração de alimentos funcionais e ser aplicados em tratamentos para pessoas com hipertensão por seu efeito inibitório sobre a ECA-I.

Relationship between angiotensin II receptor expression and cardiovascular risk factors in Mexican patients with coronary occlusive disease.

The density of Angiotensin II (Ang) receptors on tissue surfaces is regulated by multiple hormones, cytokines and metabolic factors and is profoundly affected by various pathological conditions, such as age, diet and environmental conditions. The participation of several cardiovascular risk factors in the regulation of Angiotensin II receptor expression has been incompletely studied. We performed an ex-vivo study with human aortic postsurgical specimens to test the hypothesis that Ang AT1 and AT2 receptor expression in human aortic arteries is associated with the presence of cardiovascular risk factors. We included 31 Mexican patients with coronary artery disease. We evaluated Angiotensin II receptor expression by immunostaining and angiotensin converting enzyme insertion/deletion (ACE I/D) polymorphisms by polymerase chain reaction. AT1 and AT2 receptor expression was increased in the aortic segments from the cardiovascular patients compared with control arteries and in patients with the DD genotype. There was a correlation between increased AT1 receptor expression and the number of cardiovascular risk factors present in the patient. Furthermore, reduction of AT1 expression correlated with the number of drug combinations used in the patients. These correlations were not present with respect to AT2 receptor expression. We suggest that increased AT1 receptor expression is associated with the DD genotype. Thus the presence of several cardiovascular risk factors as well as DD genotype, induce AT1 expression increasing the probability to develop coronary occlusive disease.

Hypotensive mechanism of the extracts and artemetin isolated from Achillea millefolium L. (Asteraceae) in rats.

Traditional uses of Achillea millefolium L. (Asteraceae) include the treatment of cardiovascular diseases. In the present study, we used anesthetized rats to assess the hypotensive effect of a hydroethanolic extract (HEAM), and its dichloromethane (DCM), ethyl acetate (EA), butanolic (BT), and dichloromethane-2 (DCM-2) fractions, besides the flavonoid artemetin, isolated from A. millefolium. The oral administration of HEAM (100-300 mg/kg), DCM (20mg/kg), DCM-2 (10-30 mg/kg), but not EA (10 mg/kg) and BT (50 mg/kg) fractions significantly reduced the mean arterial pressure (MAP) of normotensive rats. The phytochemical analysis by NMR (1)H of DCM and DCM-2 fractions revealed high amounts of artemetin, that was isolated and administered by either oral (1.5 mg/kg) or intravenous (0.15-1.5 mg/kg) routes in rats. This flavonoid was able to dose-dependently reduce the MAP, up to 11.47 ± 1.5 mmHg (1.5 mg/kg, i.v.). To investigate if artemetin-induced hypotension was related to angiotensin-converting enzyme inhibition, we evaluated the influence of this flavonoid on the vascular effects of both angiotensin I and bradykinin. Intravenous injection of artemetin (0.75 mg/kg) significantly reduced the hypertensive response to angiotensin I while increased the average length of bradykinin-induced hypotension. Artemetin (1.5 mg/kg, p.o.) was also able to reduce plasma (about 37%) and vascular (up to 63%) ACE activity in vitro, compared to control group. On the other hand, artemetin did not change angiotensin II-induced hypertension. Our study is the first showing the hypotensive effects induced by the extract and fractions obtained from A. millefollium. In addition, our results disclosed that this effect may be, at least in part, associated with high levels of artemetin and its ability to decrease angiotensin II generation in vivo, by ACE inhibition.

Angiotensin-converting enzyme inhibitory activity of milk fermented by wild and industrial Lactococcus lactis strains.

Angiotensin I-converting enzyme inhibitory (ACEI) activity was evaluated and compared in <3 KDa water-soluble extracts (WSE) isolated from milk fermented by wild and commercial starter culture Lactococcus lactis strains after 48 h of incubation. The highest ACEI activities were found in WSE from milk inoculated with wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures. On the other hand, the lowest ACEI activities were found in WSE from milk inoculated with wild strains isolated from vegetables. Moreover, the IC(50) values (concentration that inhibits 50% activity) of WSE from artisanal dairy products were the lowest, indicating that these fractions were the most effective in inhibiting 50% of ACE activity. In fact, a strain isolated from artisanal cheese presented the lowest IC(50) (13 µg/mL). Thus, it appears that wild L. lactis strains isolated from artisanal dairy products and commercial starter cultures showed good potential for the production of fermented dairy products with ACEI properties.

Modification of the amaranth 11S globulin storage protein to produce an inhibitory peptide of the angiotensin I converting enzyme, and its expression in Escherichia coli.

Amarantin is the predominant seed storage protein from amaranth. It shows a high content of essential amino acids, making this protein important from a nutritional viewpoint. The protein has two disulfide linked subunits: acidic and basic. Acidic subunit has the potential as a functional and nutraceutical protein, and it is structurally a good candidate for modification. In order to improve its functionality, the primary structure was modified in the third variable region of globulins 11S, by inserting four Val-Tyr antihypertensive peptides in tandem. The designed plasmid was expressed in Escherichia coli Origami (DE3) and then the expressed protein was purified. Mass spectrometry analysis was used to corroborate the identity of the protein by peptide mass fingerprinting; also, the modified peptide was fragmented and sequenced by mass spectrometry, corroborating thus the inserted residues. The hydrolyzed protein showed a high inhibitory activity of the angiotensin converting enzyme (IC(50) 0.064 mg ml(-1)); it was nearly eightfold more active than the nonmodified protein. In spite that the nonmodified subunit is less active, its activity is comparable with other hydrolyzed proteins reported as high active inhibitors. The expressed and purified subunit after its engineered modification, may be useful for preventing hypertension and for other medical purposes.