Converting Enzyme Activity Research Articles

Anti-Hypertensive Effect of a Solar Salt Diet in Elderly Hypertensive Patients: A Preliminary Randomized, Double-Blind Clinical Trial

Background: High sodium and/or low mineral intake are known to be associated with elevated blood pressure. It has been reported that substituting low-sodium, mineral-rich salt for refined salt lowers blood pressure (BP). And solar salt is emerging as a low sodium high mineral salt for a healthy diet in Korea. Therefore, this double-blind, randomized, and placebo-controlled trial was conducted to explore changes in BP from substituting refined salt with solar salt among hypertensive elderly subjects. Methods: Forty-three hypertensive and institutionalized elderly individuals aged 65 years or older were enrolled. Thirty-eight subjects (88.4%) completed the study. Subjects were provided with either a solar saltor refined salt-based diet for eight weeks. Results: Systolic BP decreased significantly in the solar salt-based diet group after 2, 4, and 8 weeks when compared to the refined salt-based diet group. And, diastolic BP was lowered significantly in the solar salt-based diet group compared to that in the refined salt-based diet group after 8 weeks. In addition, urinary sodium/potassium, and angiotension converting enzyme activity decreased significantly in the solar salt-based diet group compared to the refined salt-based group. Urinary potassium excretion was significantly increased in the solar salt-based diet group. Conclusions: These results may provide clinical evidence that solar salt has beneficial effects on BP in elderly patients. And, people such as Koreans, who do not consume enough minerals, may experience a greater anti-hypotensive effect by using solar salt. However, further large-scale studies are necessary. Korean J Health Promot 2015;15(3):98-107

Ethanol withdrawal increases oxidative stress and reduces nitric oxide bioavailability in the vasculature of rats

We analyzed the effects of ethanol withdrawal on the vascular and systemic renin-angiotensin system (RAS) and vascular oxidative stress. Male Wistar rats were treated with ethanol 3–9% (v/v) for a period of 21 days. Ethanol withdrawal was induced by abrupt discontinuation of the treatment. Experiments were performed 48 h after ethanol discontinuation. Rats from the ethanol withdrawal group showed decreased exploration of the open arms of the elevated-plus maze (EPM) and increased plasma corticosterone levels. Ethanol withdrawal significantly increased systolic blood pressure and plasma angiotensin II (ANG II) levels without an effect on plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, or plasma angiotensin I (ANG I) levels. No differences in vascular ANG I, ANG II levels, and ACE activity/expression and AT1 and AT2 receptor expression were detected among the experimental groups. Plasma osmolality, as well as plasma sodium, potassium, and glucose levels were not affected by ethanol withdrawal. Ethanol withdrawal induced systemic and vascular oxidative stress, as evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels and the vascular generation of superoxide anion. Ethanol withdrawal significantly decreased plasma and vascular nitrate/nitrite levels. Major new findings of the present study are that ethanol withdrawal induces vascular oxidative stress and reduces nitric oxide (NO) levels in the vasculature. Additionally, our study provides novel evidence that ethanol withdrawal does not affect the vascular ANG II generating system while stimulating systemic RAS. These responses could predispose individuals to the development of cardiovascular diseases.

P169 - Sodium nitrite does not inhibit circulating angiotensin converting enzyme activity

P169 - Sodium nitrite does not inhibit circulating angiotensin converting enzyme activity

Antihypertensive effect of Carica papaya via a reduction in ACE activity and improved baroreflex.

The aims of this study were to evaluate the antihypertensive effects of the standardised methanolic extract of Carica papaya, its angiotensin converting enzyme inhibitory effects in vivo, its effect on the baroreflex and serum angiotensin converting enzyme activity, and its chemical composition. The chemical composition of the methanolic extract of C. papaya was evaluated by liquid chromatography-mass/mass and mass/mass spectrometry. The angiotensin converting enzyme inhibitory effect was evaluated in vivo by Ang I administration. The antihypertensive assay was performed in spontaneously hypertensive rats and Wistar rats that were treated with enalapril (10 mg/kg), the methanolic extract of C. papaya (100 mg/kg; twice a day), or vehicle for 30 days. The baroreflex was evaluated through the use of sodium nitroprusside and phenylephrine. Angiotensin converting enzyme activity was measured by ELISA, and cardiac hypertrophy was evaluated by morphometric analysis. The methanolic extract of C. papaya was standardised in ferulic acid (203.41 ± 0.02 µg/g), caffeic acid (172.60 ± 0.02 µg/g), gallic acid (145.70 ± 0.02 µg/g), and quercetin (47.11 ± 0.03 µg/g). The flavonoids quercetin, rutin, nicotiflorin, clitorin, and manghaslin were identified in a fraction of the extract. The methanolic extract of C. papaya elicited angiotensin converting enzyme inhibitory activity. The antihypertensive effects elicited by the methanolic extract of C. papaya were similar to those of enalapril, and the baroreflex sensitivity was normalised in treated spontaneously hypertensive rats. Plasma angiotensin converting enzyme activity and cardiac hypertrophy were also reduced to levels comparable to the enalapril-treated group. These results may be associated with the chemical composition of the methanolic extract of C. papaya, and are the first step into the development of a new phytotherapic product which could be used in the treatment of hypertension.

Dohaekseunggi-tang extract inhibits obesity, hyperlipidemia, and hypertension in high-fat diet-induced obese mice.

BackgroundDohaekseunggi-tang (DHSGT) is a traditional plant-based medicine prescribed to promote blood circulation and to treat obesity and hypertension. The present study aimed to identify potential anti-obesity activities of DHSGT extract.MethodsAnti-obesity, anti-hyperlipidemic, and anti-hypertensive effects of orally-administered DHSGT extract were evaluated in high-fat diet- (HFD)-induced obese mice. Serum biochemistry profiles and expression of diverse metabolic regulatory gene mRNAs in mouse visceral fat were assessed by RT-PCR. The effects of DHSGT on angiotensin-1 converting enzyme (ACE) and pancreatic lipase activities were determined using in vitro inhibition assays.ResultsOral DHSGT treatment reduced obese HFD C57BL/6 J mouse body weight, liver and adipose tissue mass, adipocyte size, and blood pressure versus untreated HFD mice. DHSGT also decreased serum total cholesterol, LDL-cholesterol, triglyceride, glucose, and leptin concentrations, and increased HDL-cholesterol and adiponectin levels in HFD mice. Furthermore, DHSGT markedly increased mRNA expression of peroxisome proliferator activated receptor-gamma, uncoupling protein-2, and adiponectin in visceral adipose tissue of HFD mice. In vitro tests revealed that DHSGT effectively inhibited porcine pancreatic lipase and ACE activities, with IC50 values of 7.58 mg/ml and 0.56 mg/ml, respectively.ConclusionsThese results validate traditional knowledge and suggest that DHSGT may be potentially useful for managing hyperlipidemia, hyperglycemia, hypertension, and obesity.

Angiotensin I Converting Enzyme (ACE) inhibitory activity and antihypertensive effects of grass carp peptides

In vitro and in vivo antihypertensive effects of grass carp peptides (GCP) were investigated. The amino acid composition and the angiotensinconverting enzyme inhibitory activity were evaluated using HPLC. GCP was administrated to spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKY) by single and long-term administration before blood pressure measurements. Plasma levels of angiotensin II (Ang II), rennin (RA), nitric oxide (NO), and plasma angiotensin I converting enzyme (ACE) activity were measured. The GCP molecular weight was between 725 and 1,228 Da with high levels of Leu, Asp, Phe, Gly, and Pro. The in vitro IC50 value was 0.23 mg/mL. GCP lowered SHR blood pressure dose and timedependently. Little change occurred in WKY. The plasma level of Ang II and the ACE activity decreased. SHR RA and NO concentrations in plasma increased. The antihypertensive effect of GCP was associated with NO regulation and the rennin-angiotensin system.

Abstract 2906: An effective anti-EpCAM antibody EpAb2-6 for the treatment of colon cancer and its underlying mechanisms

Abstract Epithelial cell adhesion molecule (EpCAM) is known to be overexpressed in epithelial cancers associated with enhanced malignant potential, particularly colorectal carcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC). However, it is yet to be determined whether targeting EpCAM can directly inhibit the progression of malignance. Here, we have generated six novel monoclonal antibodies (mAbs) against EpCAM; these antibodies specifically bind to CRC and HNSCC cells, but not normal cells. One EpCAM mAb, EpAb2-6, was found to induce cancer cell apoptosis in vitro, inhibit tumor growth, and prolong the overall survival of mice with human colon or oral carcinoma xenografts. EpAb2-6 also increases the therapeutic efficacy of irinotecan, fluorouracil, and leucovorin (IFL) therapy in tumor-bearing mice. Subsequent analysis suggested that EpAb2-6 can potentially inhibit the growth and proliferation of tumorspheres. Investigation of the mechanism by which EpAb2-6 inhibits tumor growth revealed that EpAb2-6 inhibits tumor-necrosis-factor α converting enzyme (TACE/ADAM17) and γ-secretase activation and blocks EpCAM intracellular domain (EpICD) cleavage, thus preventing the nuclear localization of EpICD. Furthermore, EpAb2-6, which binds to positions in the EGF-II/TY domain of EpCAM, inhibits production of EpICD, thereby decreasing translocation of β-catenin into the nucleus and activation of p53 and p21 expression. Collectively, our results indicate that novel EpCAM mAbs can potentially be used for cancer-targeted therapy. Citation Format: Han-Chung Wu, Yi-Hsuan Chi, Mei-Ying Liao. An effective anti-EpCAM antibody EpAb2-6 for the treatment of colon cancer and its underlying mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2906. doi:10.1158/1538-7445.AM2014-2906

Comparison of the Effects of Matrix Metalloproteinase Inhibitors on TNF-α Release from Activated Microglia and TNF-α Converting Enzyme Activity.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that regulate cell-matrix composition and are also involved in processing various bioactive molecules such as cell-surface receptors, chemokines, and cytokines. Our group recently reported that MMP-3, -8, and -9 are upregulated during microglial activation and play a role as proinflammatory mediators (Lee et al., 2010, 2014). In particular, we demonstrated that MMP-8 has tumor necrosis factor alpha (TNF-α)-converting enzyme (TACE) activity by cleaving the prodomain of TNF-α and that inhibition of MMP-8 inhibits TACE activity. The present study was undertaken to compare the effect of MMP-8 inhibitor (M8I) with those of inhibitors of other MMPs, such as MMP-3 (NNGH) or MMP-9 (M9I), in their regulation of TNF-α activity. We found that the MMP inhibitors suppressed TNF-α secretion from lipopolysaccharide (LPS)-stimulated BV2 microglial cells in an order of efficacy: M8I>NNGH>M9I. In addition, MMP inhibitors suppressed the activity of recombinant TACE protein in the same efficacy order as that of TNF-α inhibition (M8I>NNGH>M9I), proving a direct correlation between TACE activity and TNF-α secretion. A subsequent pro-TNF-α cleavage assay revealed that both MMP-3 and MMP-9 cleave a prodomain of TNF-α, suggesting that MMP-3 and MMP-9 also have TACE activity. However, the number and position of cleavage sites varied between MMP-3, -8, and -9. Collectively, the concurrent inhibition of MMP and TACE by NNGH, M8I, or M9I may contribute to their strong anti-inflammatory and neuroprotective effects.

Comparison of the Effects of Matrix Metalloproteinase Inhibitors on TNF-a Release from Activated Microglia and TNF-a Converting Enzyme Activity

Comparison of the Effects of Matrix Metalloproteinase Inhibitors on TNF-a Release from Activated Microglia and TNF-a Converting Enzyme Activity

Phenolic compounds from sandpaper (ficus exasperata) leaf inhibits angiotensin 1 converting enzyme in high cholesterol diet fed rats

Ethnopharmacological relevanceSandpaper [Ficus exasperata Vahl (Moraceae)] leaf has been reportedly used in folklore for the management/treatment of cardiovascular diseases with little/or no scientific basis for their action. This study sought to investigate the effect of dietary supplementation of sandpaper leaf on angiotensin-I converting enzyme (ACE) activity in hypercholesterolemia as well as the effect of their phenolic extract on this enzyme in vitro. Materials and methodsThe phenolic extract was prepared, then, the inhibitory effect of the leaf extract on ACE was determined in vitro. Thereafter, the effect of dietary supplementation of sandpaper leaf on angiotensin-I converting enzyme (ACE) activity in high cholesterol diet fed rats for 14 days was evaluated as well as some biochemical parameters. ResultsThe result revealed that under in vitro condition, the phenolic extract inhibited ACE (IC50=14.7µg/mL) in a dose-dependent manner (0–10µg/mL). Feeding high cholesterol diets to rats caused a significant (P<0.05) increase in the ACE activity. However, there was a significant (P<0.05) decrease in the ACE activity as a result of supplementation with the sand paper leaves. Furthermore, there was a significant (P<0.05) increase in the plasma lipid profile with a concomitant increase in malondialdehyde (MDA) content in rat liver and heart tissues. However, supplementing the diet with sandpaper leaf (either 10% or 20%) caused a significant (P<0.05) decrease in the plasma total cholesterol (TC), triglyceride (TG), very low density lipoprotein-cholesterol (VLDL-C), and low-density lipoprotein-cholesterol levels (LDL-C), and in MDA content in the tissues. Conversely, supplementation caused a significant (P<0.05) increase in plasma high-density lipoprotein-cholesterol level when compared with the control diet. Reversed phase HPLC analysis of the extract revealed Quercitrin (43.7mg/g), chlorogenic acid (42.8mg/g) and caffeic acid (33.9mg/g) as the major phenolics in the leaf. ConclusionThe inhibition of ACE activity and prevention of hypercholesterolemia by sandpaper leaf could be part of the possible mechanism underlying its anti-hypertensive property which could lay credence to its use in folk medicine. However, these activities may be directly/indirectly attributed to the polyphenolics present.

In Vitro Studies on the Antioxidant Property and Inhibition of α-Amylase, α-Glucosidase, and Angiotensin I-Converting Enzyme by Polyphenol-Rich Extracts from Cocoa (Theobroma cacao) Bean.

Background. This study sought to investigate the antidiabetic and antihypertensive mechanisms of cocoa (Theobroma cacao) bean through inhibition of α-amylase, α-glucosidase, angiotensin-1 converting enzyme, and oxidative stress. Methodology. The total phenol and flavonoid contents of the water extractable phytochemicals from the powdered cocoa bean were determined and the effects of the extract on α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities were investigated in vitro. Furthermore, the radicals [1,1-diphenyl-2 picrylhydrazyl (DPPH), 2,2..-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), hydroxyl (OH), and nitric oxide (NO)] scavenging ability and ferric reducing antioxidant property of the extract were assessed. Results. The results revealed that the extract inhibited α-amylase (1.81 ± 0.22 mg/mL), α-glucosidase (1.84 ± 0.17 mg/mL), and angiotensin-1 converting enzyme (0.674 ± 0.06 mg/mL [lungs], 1.006 ± 0.08 mg/mL [heart]) activities in a dose-dependent manner and also showed dose-dependent radicals [DPPH (16.94 ± 1.34 mg/mL), NO (6.98 ± 0.886 mg/mL), OH (3.72 ± 0.26 mg/mL), and ABTS (15.7 ± 1.06 mmol/TEAC·g] scavenging ability. Conclusion. The inhibition of α-amylase, α-glucosidase, and angiotensin-1 converting enzyme activities by the cocoa bean extract could be part of the possible mechanism by which the extract could manage and/or prevent type-2 diabetes and hypertension.

Matrix metalloproteinase-8 plays a pivotal role in neuroinflammation by modulating TNF-α activation.

Matrix metalloproteinases (MMPs) play important roles in normal brain development and synaptic plasticity, although aberrant expression of MMPs leads to brain damage, including blood-brain barrier disruption, inflammation, demyelination, and neuronal cell death. In this article, we report that MMP-8 is upregulated in LPS-stimulated BV2 microglial cells and primary cultured microglia, and treatment of MMP-8 inhibitor (M8I) or MMP-8 short hairpin RNA suppresses proinflammatory molecules, particularly TNF-α secretion. Subsequent experiments showed that MMP-8 exhibits TNF-α-converting enzyme (TACE) activity by cleaving the prodomain of TNF-α (A(74)/Q(75), A(76)/V(77) residues) and, furthermore, that M8I inhibits TACE activity more efficiently than TAPI-0, a general TACE inhibitor. Biochemical analysis of the underlying anti-inflammatory mechanisms of M8I revealed that it inhibits MAPK phosphorylation, NF-κB/AP-1 activity, and reactive oxygen species production. Further support for the proinflammatory role of microglial MMP-8 was obtained from an in vivo animal model of neuroinflammatory disorder. MMP-8 is upregulated in septic conditions, particularly in microglia. Administration of M8I or MMP-8 short hairpin RNA significantly inhibits microglial activation and expression/secretion of TNF-α in brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice. These results demonstrate that MMP-8 critically mediates microglial activation by modulating TNF-α activity, which may explain neuroinflammation in septic mouse brain.

Triterpenes from the Protium heptaphyllum resin – chemical composition and cytotoxicity

Protium heptaphyllum (Aubl) Marchand, Burseraceae, is popularly used as an analgesic and anti-inflammatory agent. However, the cellular mechanism of action remains unknown. This study aims to evaluate the chemical composition of P. heptaphyllum resin and cytotoxicity on a breast cancer cell line (MCF-7). The chemical composition of the resin was determined by Gas Chromatography coupled to a Mass Spectrometer. The cytotoxicity was evaluated using an MTT assay. Annexin V-FITC, caspase-3, Angiotensin Converting Enzyme activity and Tumor Necrosis Factor alpha (TNF- α) assays were performed to evaluate apoptosis and inflammatory events. The resin consisted of triterpenes, such as α- and β-amyrin. Cytotoxicity was only observed in fractions enriched with α- and β-amyrin. The resin and fractions elicited antiproliferative activity, increased activity of caspase-3 and ACE, and a decrease in the TNF-α level. Altogether, the resin and fractions enriched with α- and β-amyrin promoted cytotoxicity and apoptosis.

복분자 추출물의 항고혈압 활성

본 연구는 복분자 완숙과 50% 에탄올 추출물을 이용하여 인간제대정맥내피세포주(HUVEC)에서 복분자 추출물이 NO의 생성에 미치는 영향과 ACE 단백질 발현을 조사하였으며, 본태성 고혈압 흰쥐(SHR)에서 혈압강하 효과 및 혈압의 조절과 관련 깊은 인자들(renin, AT1-R, ET-1, IL-6)의 유전자 발현 그리고 혈관조직의 변화를 조사하였다. 그 결과 복분자 완숙과 50% 에탄올 추출물은 HUVEC 세포주에서 NO의 생성을 촉진하였으며 혈관수축활성을 가지는 ACE 단백질의 발현을 억제하였고 본태성 고혈압 흰쥐에게 복분자 완숙과 에탄올 50% 추출물을 8주간 투여한 결과 혈액 내 ACE의 활성이 억제되었으며 renin 및 혈관 수축인자인 ET-1 유전자의 발현이 억제되고 염증관련 유전자인 IL-6의 발현이 억제됨을 확인하였다. 그리고 신세동맥의 두께 및 심근 호산성세포수를 감소시켜 혈관내피세포의 손상을 보호하며 심장의 병변을 경감시키는 효과가 있음을 확인하였다. 본 연구 결과 복분자 완숙과 추출물은 혈압 조절과 연관된 단백질 및 유전자의 발현과 활성을 조절하고, 혈관의 두께 및 심근의 변성을 감소시켜 혈압 강하에 도움이 되는 것으로 나타났으며 향후 심혈관계 질환을 개선시킬 수 있는 식품소재로서의 가능성이 있음을 확인하였다. This study aimed to investigate the effects of 50% ethanol extract of ripe black raspberry (Rubus occidentalis, RBR) on hypertension in human umbilical vein endothelial cells (HUVECs) and in spontaneously hypertensive rats (SHR). Angiotensin converting enzyme (ACE) inhibition and activation of nitric oxide production by endothelial nitric oxide synthase were significantly regulated by RBR in HUVEC cells. Moreover, the SHR showed significantly higher levels of blood pressure, ACE, renin, endothelin-1, and interleukin-6 than Wistar Kyoto rats (WKY). However, treatment with captopril and RBR decreased the levels of these hypertension-related events in the SHR. The renal arteriole showed greater media thickness/lumen diameter (%) in the SHR than in the WKY. However, media thickness/lumen diameter (%) was reduced in SHR by treatment with captopril and RBR. In addition, the number of eosinophilic cardiac muscle cells was decreased in the heart muscles after treatment with captopril and RBR. Therefore, this study suggests that 50% ethanol extract of RBR may be useful for the prevention and treatment of high blood pressure.

The antihypertensive effects of sodium nitrite are not associated with circulating angiotensin converting enzyme inhibition

Nitrite-derived nitric oxide (NO) formation exerts antihypertensive effects. Because NO inhibits angiotensin converting enzyme (ACE) activity, we carried a comprehensive series of experiments in rats to test the hypothesis that sodium nitrite exerts antihypertensive effects by inhibiting ACE. We examined whether sodium nitrite (15mg/kg; or vehicle; by gavage): (I) attenuates the pressor responses to angiotensin I at doses of 0.03, 0.1, 0.3, 1, 3, and 10μg/kg intravenously; (II) attenuates the acute hypertension induced by L-NAME (100mg/kg; or vehicle; by gavage); (III) attenuates the chronic hypertension induced by L-NAME (1g/L in drinking water; or vehicle) administered for 6weeks; (IV) attenuates the hypertension in the 2 kidney-1 clip (2K1C) chronic hypertension model. Blood samples were collected at the end of each study and plasma angiotensin converting enzyme (ACE) activity was measured with a fluorimetric assay using Hippuryl-His-Leu as substrate. ACE inhibitors were used as positive controls. Plasma nitrite concentrations were measured by ozone-based reductive chemiluminescence. The in vitro effects of sodium nitrite (0, 1, 3, 10, 30, 100μmol/L) on plasma ACE activity were also determined. We found that sodium nitrite did not affect the pressor responses to angiotensin I. Moreover, while sodium nitrite exerted significant antihypertensive effects in acute and chronic hypertension models, no significant effects on plasma ACE activity were found. In vitro experiments showed no effects of sodium nitrite on plasma ACE activity. This is the first study to demonstrate that the acute and chronic antihypertensive effects of sodium nitrite are not associated with significant inhibition of circulating ACE activity.

Cardiovascular protective effects of Casearia sylvestris Swartz in Swiss and C57BL/6 LDLr-null mice undergoing high fat diet

Ethnopharmacological relevanceAlthough Casearia sylvestris Swartz is used in Brazilian folk medicine to treat obesity, no study has been conducted to evaluate the effects of this species in an experimental model of dyslipidemia and atherosclerosis. So, the aim of this study was to evaluate possible hypolipemiant and antiatherogenic activity of the methanolic extract obtained from Casearia sylvestris (MECS) using Swiss and C57BL/6 LDLr-null mice undergoing high fat diet (HFD). Material and methodsDyslipidemia and atherogenesis were induced by the administration of commercial HFD for 4 weeks. The MECS was administered orally at doses of 250 and 500mg/kg, once a day, for two weeks, starting from the 2nd week of HFD. The gain in body weight and systolic blood pressure (SBP) were measured weekly over the four week study. At the end of the experiments the levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C) were measured by colorimetric method. Aldosterone, vasopressin and angiotensin converting enzyme (ACE) activity were also evaluated in collected serum. The renal function, atherogenic index serum (AIS) and in vitro antiplatelet activity were investigated. Additionally, histopathological analyzes were performed to determine the intima-media thickness (IMT) and intima media ratio (IMR) in aorta samples. ResultsThe HFD induced dyslipidemia and major structural changes in the aortic wall, including raising of the systolic blood pressure in LDLr-null mice. In addition, we observed an increase in lipid peroxidation accompanied by a reduction of serum nitrite. The treatment with MECS was able to prevent the increase of SBP, TC, LDL-C, VLDL-C and triglycerides levels and increase HDL-C in Swiss and LDLr-null mice. These effects were accompanied by a significant reduction in oxidative stress. Moreover, AIS, IMT and IMR were significantly reduced in MECS-treated mice, and the extract was able to reduce platelet aggregation in vitro. ConclusionThis study demonstrated that MECS reduces the serum lipids and oxidative stress when orally administered to Swiss and LDLr-null mice. In addition, it was able to prevent arterial thickening induced by HFD and to inhibit platelet aggregation in vitro.

Anti-septic Effects of Fisetin In Vitro and In Vivo

Sepsis is a state of disrupted inflammatory homeostasis that is initiated by infection. High mobility group box 1 (HMGB1) protein acting as a late mediator of severe vascular inflammatory conditions, such as sepsis and endothelial cell protein C receptor (EPCR), is involved in vascular inflammation. Fisetin, an active compound from the family Fabaceae, was reported to have antiviral, neuroprotective, and anti-inflammatory activities. Here, we determined the anti-septic effects of fisetin on HMGB1-mediated inflammatory responses and on the shedding of EPCR in vitro and in vivo, for the first time. First, we monitored the effects of post-treatment fisetin on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Post-treatment fisetin was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. Fisetin also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. Fisetin induced potent inhibition of phorbol-12-myristate 13-acetate (PMA) and CLP-induced EPCR. Fisetin also inhibited the expression and activity of tumor necrosis factor-α converting enzyme, induced by PMA in endothelial cells. In addition, fisetin inhibited the production of tumor necrosis factor-α and the activation of AKT, nuclear factor-κB, and extracellular regulated kinases 1/2 by HMGB1 in HUVECs. Fisetin also down-regulated CLP-induced release of HMGB1, production of interleukin 1β, and reduced septic mortality. Collectively, these results suggest that fisetin may be a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

Development of a high-resolution melting genotyping assay for the angiotensin I converting enzyme insertion/deletion polymorphism and establishment of genotype-specific reference intervals in a Danish population

The serum-angiotensin I converting enzyme (s-ACE) activity is influenced by a genetic insertion/deletion (I/D) polymorphism in the ACE gene, and the resulting large interindividual variation in s-ACE limits the use of normal reference intervals in the evaluation of sarcoidosis. In this study, we developed a new method for genotyping the I/D polymorphism in ACE and established genotype-specific reference intervals in order to improve the diagnostic accuracy and the value for treatment of sarcoidosis. The new genotyping assay is based on high-resolution melting (HRM) using LCGreen + and was used to genotype 400 healthy Danish individuals. The assay was compared to a real-time polymerase chain reaction (RT-PCR) assay in a validation set of 86 samples. Enzyme activity in serum was measured using the Infinity™ ACE Liquid Stable Reagent from Thermo adapted for the ABX Pentra analyzer. There was full concordance between genotyping assays. The three genotypes II, ID and DD were present with a frequency of 0.23, 0.51 and 0.26. The distribution of s-ACE values in the total population was non-Gaussian (non-parametric 95% reference interval 12.0-60.0 U/L). The median activities of the genotypes differed significantly (P<0.0001). Ninety-five per cent non-parametric reference intervals for the subpopulations were determined to 6.3-38.5, 14.0-56.0 and 23.3-71.2 U/L for II, ID and DD, respectively. We have developed a simple and robust method for ACE genotyping and determined genotype-specific reference intervals for s-ACE concentrations in the Danish population. The new reference intervals may increase the value of s-ACE measurements.

Aqueous Garlic Extract Alleviates Liver Fibrosis and Renal Dysfunction in Bile-Duct-Ligated Rats

There is accumulating evidence that the renin-angiotensin system (RAS) is involved in hepatic inflammation and fibrogenesis. Garlic was found to lower the activity of the angiotensin converting enzyme (ACE) in the serum of rats in a diabetic model. We examined the effect of an aqueous garlic extract (AGE) on the ACE activity, cholestasis-induced liver fibrosis, and associated renal dysfunction in comparison with the effect of the standard drug enalapril. Both AGE and enalapril were administered orally for six weeks starting from the third day after bile duct ligation (BDL). BDL significantly increased the serum activities of liver enzymes, serum lactate dehydrogenase (LDH) activity, an indicator of liver cell death, serum total bilirubin (TB) level, liver myeloperoxidase (MPO) activity, and liver malondialdehyde (MDA) content. BDL was associated with elevation of serum urea and creatinine levels indicating renal dysfunction. BDL also caused an increase in the transcript levels of the genes coding for tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1), and matrix metalloproteinase-13 (MMP-13), a collagenase, in liver tissues. A significant decrease in hepatic reduced glutathione (GSH) was observed in BDL rats, while serum ACE activity was increased. Both AGE and enalapril counteracted all these deleterious changes, with the exception that only AGE reduced the MPO activity. These findings suggest that AGE possesses hepato- and renoprotective properties, similar to enalapril, probably by modulating the levels of proteins such as TNF-alpha, TGF-beta1 and MMP-13, and involving a reduction of ACE and of oxidative stress.

Bioactive peptides produced from enhanced common bean cultivars inhibit angiotensin converting enzyme activity (647.14)

Common bean (Phaseolus vulgaris L.) is a good source of proteins (16‐33%), vitamins, minerals and complex carbohydrates. The objective was to produce, characterize and evaluate the angiotensin converting enzyme (ACE) inhibition and antioxidant capacity (AC) of bioactive peptides from proteins of common bean cultivars from Mexico and Brazil. Bean protein isolates were hydrolyzed by pepsin‐pancreatin and the molecular mass of the produced peptides determined by mass spectrometry. Phaseolin and lectin were the major proteins identified in the studied common beans. Forty seven abundant peptides were identified ranging from 347 to 3067 Da in beans BRS‐Horizonte, BRS‐ Pontal, Perola and Black‐Otomi; the black bean presented the highest amount of identified peptides (689.3, 713.4, 959.6, 981.6 and 1275.7 Da). Common bean Pontal showed the highest AC value (274 mmol ± 0.8 TE/g protein) and BRS‐Horionte the lowest (240 ± 3.8 mmol TE/g protein). ACE inhibition by peptides from Black‐Otomi (YG, RP, GT, GG, DG, NY, CF) was the highest (IC50 = 0.29 ± 0.08 mg /mL) and Pontal the lowest (IC50 = 0.45 ± 0.06 mg/mL), p &lt; 0.05. Mexico and Brazil cultivars showed similar peptide pattern. Peptides from common bean showed high ACE inhibitor activity, high AC, and potential to enhance health and wellness if used as functional ingredients.Grant Funding Source: Supported by CONACYT, Research Council for Science and Technology