Tratamientos y bioterapias para COVID-19 / Therapeutics and biotherapies for covid-19

INTRODUCCIÓN: Un nuevo brote de coronavirus surgió en 2019 en Wuhan, China, causando conmoción en el sistema sanitario de todo el mundo; el Comité Internacional de Taxonomía de Virus lo denominó SARS-CoV-2, agente causante de la enfermedad COVID-19.El espectro de gravedad de la enfermedad es muy amplio: la mayoría de los pacientes no presentan gravedad, pero otros pueden desarrollar neumonías, y la insuficiencia respiratoria aguda es la causa más frecuente de mortalidad. Objetivo: analizar y desarrollar las distintas alternativas terapéuticas aportadas por la Biotecnología para tratar los síntomas de aquellos pacientes con COVID-19. Metodología: se realizó una revisión de la bibliografía disponible, a partir de enero de 2020 en PubMed, acerca de los tratamientos que se encuentran aún en ensayos clínicos y aquellos que cuentan con aprobación bajo uso de emergencia para la enfermedad COVID-19. También se realizaron búsquedas a través de Google y Google Académico para publicaciones de organismos de Salud en referencia a políticas de salud establecidas para la terapéutica durante dicha pandemia. Resultados: este trabajo aborda las nuevas alternativas terapéuticas para COVID-19 derivadas de la Biotecnología, que se encuentran tanto en uso como en etapas de ensayos clínicos comprendidos dentro del segmento de los biofármacos y las bioterapias. Se incluye un breve resumen del estatus regulatorio de entidades de salud, el mecanismo de acción de dichas terapias y características generales de cada uno. Se incluyen novedosas bioterapias que se empezaron a implementar para afrontar la pandemia. Conclusiones: la pandemia de coronavirus está poniendo a prueba el sistema sanitario internacional, para brindar soluciones tanto desde el diagnóstico y prevención como para el tratamiento de la población a fin de disminuir la mortalidad. Esto incluyó, obviamente también, al área de la Biotecnología aplicada a la salud, que ha aportado en los tres aspectos mencionados; el presente trabajo se centra en las respuestas de tipo terapéutico que ha brindado y que están comercializadas o en fases clínicas. (AU)

INTRODUCTION: A new coronavirus outbreak emerged in 2019 in Wuhan, China, causing a shock to the healthcare system around the world; the International Committee on Taxonomy of Viruses named it SARS-CoV- 2, the infectious agent of the COVID-19 disease. The spectrum of severity of the disease is very wide, most patients are not serious, but others can develop pneumonia, with acute respiratory failure being the most frequent cause of mortality. Objective: to analyze and develop the different therapeutic alternatives provided by Biotechnology dedicated to Health, to treat the symptoms of those COVID-19 patients who require it, and thus reduce mortality.Methodology: a review of the available literature from January 2020 in PubMed of the treatments that are still in clinical trials and those that have been approved under emergency use for the disease COVID-19 was performed. Searches were also carried out through Google and Google Scholar for publications of Health organizations in reference to health policies established for therapeutics during the mentioned pandemic. Results: this work addresses the new therapeutic alternatives derived from Biotechnology, which are both in use and in stages of clinical trials, to treat patients who developed COVID-19 included within the segment of biopharmaceuticals and biotherapies. A brief summary of the regulatory status of health entities, the mechanism of action of said therapies and general characteristics of each one is included. Innovative biotherapies that began to be implemented to face the pandemic are included. Conclusions: The coronavirus pandemic has driven the international health system to the test, to provide solutions both from the diagnosis, prevention and treatment of the population to reduce the mortality of patients. This obviously also included the area of Biotechnology applied to health, which has contributed in the three aspects mentioned. The present work focuses on the therapeutic responses that it has provided and that are commercialized or in clinical phases. (AU)

Glycyrrhizin: An old weapon against a novel coronavirus.

Currently, over 100 countries are fighting against a common enemy, the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, which causes COVID-19. This has created a demand for a substance whose effectiveness has already been demonstrated in a similar scenario. Glycyrrhizin (GZ) is a promising agent against SARS-CoV-2 as its antiviral activity against SARS-CoV has already been confirmed. It is worthwhile to extrapolate from its proven therapeutic effects as there is a high similarity in the structure and genome of SARS-CoV and SARS-CoV-2. There are many possible mechanisms through which GZ acts against viruses: increasing nitrous oxide production in macrophages, affecting transcription factors and cellular signalling pathways, directly altering the viral lipid-bilayer membrane, and binding to the ACE2 receptor. In this review, we discuss the possible use of GZ in the COVID-19 setting, where topical administration appears to be promising, with the nasal and oral cavity notably being the potent location in terms of viral load. The most recently published papers on the distribution of ACE2 in the human body and documented binding of GZ to this receptor, as well as its antiviral activity, suggest that GZ can be used as a therapeutic for COVID-19 and as a preventive agent against SARS-CoV-2.

Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus.

BACKGROUND: The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating . The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial. PURPOSE: The objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection. METHODS: In our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2h cells. RESULTS: Results showed that HCQ is slightly more toxic to ACE2h cells than CQ. Both CQ and HCQ could bind to ACE2 with KD = (7.31 ± 0.62)e-7 M and (4.82 ± 0.87)e-7 M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2h cells. CONCLUSIONS: CQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection.

Lemon Extract Reduces Angiotensin Converting Enzyme (ACE) Expression and Activity and Increases Insulin Sensitivity and Lipolysis in Mouse Adipocytes.

Obesity is associated with insulin resistance and cardiovascular complications. In this paper, we examine the possible beneficial role of lemon juice in dieting. Lemon extract (LE) has been proposed to improve serum insulin levels and decrease angiotensin converting enzyme (ACE) activity in mouse models. ACE is also a biomarker for sustained weight loss and ACE inhibitors improve insulin sensitivity in humans. Here, we show that LE impacts adipose tissue metabolism directly. In 3T3-L1 differentiated adipocyte cells, LE improved insulin sensitivity as evidenced by a 3.74 ± 0.54-fold increase in both pAKT and GLUT4 levels. LE also induced lipolysis as demonstrated by a 16.6 ± 1.2 fold-change in pHSL protein expression levels. ACE gene expression increased 12.0 ± 0.1 fold during differentiation of 3T3-L1 cells in the absence of LE, and treatment with LE decreased ACE gene expression by 80.1 ± 0.5% and protein expression by 55 ± 0.37%. We conclude that LE's reduction of ACE expression causes increased insulin sensitivity and breakdown of lipids in adipocytes.

Alkamides and Piperamides as Potential Antivirals against the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has quickly spread globally, infecting millions and killing hundreds of thousands of people. Herein, to identify potential antiviral agents, 97 natural amide-like compounds known as alkamides and piperamides were tested against SARS-CoV-2 main protease (Mpro) and RNA-dependent RNA polymerase (RdRp), and the human angiotensin-converting enzyme 2 (ACE2) using molecular docking and molecular dynamics simulations. The docking results showed that alkamides and dimeric piperamides from Piper species have a high binding affinity and potential antiviral activity against SARS-CoV-2. The absorption, distribution, metabolism, and excretion (ADME) profile and Lipinski's rule of five showed that dimeric piperamides have druglikeness potential. The molecular dynamics results showed that pipercyclobutanamide B forms a complex with Mpro at a similar level of stability than N3-I. Our overall results indicate that alkamides and piperamides, and specifically pipercyclobutanamide B, should be further studied as compounds with SARS-CoV-2 antiviral properties.

In Silico Screening of Potential Spike Glycoprotein Inhibitors of SARS-CoV-2 with Drug Repurposing Strategy.

OBJECTIVE: To select potential molecules that can target viral spike proteins, which may potentially interrupt the interaction between the human angiotension-converting enzyme 2 (ACE2) receptor and viral spike protein by virtual screening. METHODS: The three-dimensional (3D)-coordinate file of the receptor-binding domain (RBD)-ACE2 complex for searching a suitable docking pocket was firstly downloaded and prepared. Secondly, approximately 15,000 molecular candidates were prepared, including US Food and Drug Administration (FDA)-approved drugs from DrugBank and natural compounds from Traditional Chinese Medicine Systems Pharmacology (TCMSP), for the docking process. Then, virtual screening was performed and the binding energy in Autodock Vina was calculated. Finally, the top 20 molecules with high binding energy and their Chinese medicine (CM) herb sources were listed in this paper. RESULTS: It was found that digitoxin, a cardiac glycoside in DrugBank and bisindigotin in TCMSP had the highest docking scores. Interestingly, two of the CM herbs containing the natural compounds that had relatively high binding scores, Forsythiae fructus and Isatidis radix, are components of Lianhua Qingwen (), a CM formula reportedly exerting activity against severe acute respiratory syndrome (SARS)-Cov-2. Moreover, raltegravir, an HIV integrase inhibitor, was found to have a relatively high binding score. CONCLUSIONS: A class of compounds, which are from FDA-approved drugs and CM natural compounds, that had high binding energy with RBD of the viral spike protein. Our work provides potential candidates for other researchers to identify inhibitors to prevent SARS-CoV-2 infection, and highlights the importance of CM and integrative application of CM and Western medicine on treating COVID-19.

In silico ADMET and molecular docking study on searching potential inhibitors from limonoids and triterpenoids for COVID-19.

Virtual screening of phytochemicals was performed through molecular docking, simulations, in silico ADMET and drug-likeness prediction to identify the potential hits that can inhibit the effects of SARS-CoV-2. Considering the published literature on medicinal importance, 154 phytochemicals with analogous structure from limonoids and triterpenoids were selected to search potential inhibitors for the five therapeutic protein targets of SARS-CoV-2, i.e., 3CLpro (main protease), PLpro (papain-like protease), SGp-RBD (spike glycoprotein-receptor binding domain), RdRp (RNA dependent RNA polymerase) and ACE2 (angiotensin-converting enzyme 2). The in silico computational results revealed that the phytochemicals such as glycyrrhizic acid, limonin, 7-deacetyl-7-benzoylgedunin, maslinic acid, corosolic acid, obacunone and ursolic acid were found to be effective against the target proteins of SARS-CoV-2. The protein-ligand interaction study revealed that these phytochemicals bind with the amino acid residues at the active site of the target proteins. Therefore, the core structure of these potential hits can be used for further lead optimization to design drugs for SARS-CoV-2. Also, the medicinal plants containing these phytochemicals like licorice, neem, tulsi, citrus and olives can be used to formulate suitable therapeutic approaches in traditional medicines.

Network pharmacology and molecular docking analysis on molecular targets and mechanisms of Huashi Baidu formula in the treatment of COVID-19.

PURPOSE: Huashi Baidu formula (HSBDF) was developed to treat the patients with severe COVID-19 in China. The purpose of this study was to explore its active compounds and demonstrate its mechanisms against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through network pharmacology and molecular docking. METHODS: All the components of HSBDF were retrieved from the pharmacology database of TCM system. The genes corresponding to the targets were retrieved using UniProt and GeneCards database. The herb-compound-target network was constructed by Cytoscape. The target protein-protein interaction network was built using STRING database. The core targets of HSBDF were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The main active compounds of HSBDF were docked with SARS-CoV-2 and angiotensin converting enzyme II (ACE2). RESULTS: Compound-target network mainly contained 178 compounds and 272 corresponding targets. Key targets contained MAPK3, MAPK8, TP53, CASP3, IL6, TNF, MAPK1, CCL2, PTGS2, etc. There were 522 GO items in GO enrichment analysis (p < .05) and 168 signaling pathways (p < .05) in KEGG, mainly including TNF signaling pathway, PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, and HIF-1 signaling pathway. The results of molecular docking showed that baicalein and quercetin were the top two compounds of HSBDF, which had high affinity with ACE2. CONCLUSION: Baicalein and quercetin in HSBDF may regulate multiple signaling pathways through ACE2, which might play a therapeutic role on COVID-19.

Non-steroidal anti-inflammatory drugs, pharmacology, and COVID-19 infection.

Non-steroidal anti-inflammatory drugs (NSAIDs) have an optional prescription status that has resulted in frequent use, in particular for the symptomatic treatment of fever and non-rheumatic pain. In 2019, a multi-source analysis of complementary pharmacological data showed that using NSAIDs in these indications (potentially indicative of an underlying infection) increases the risk of a severe bacterial complication, in particular in the case of lung infections. First, the clinical observations of the French Pharmacovigilance Network showed that severe bacterial infections can occur even after a short NSAID treatment, and even if the NSAID is associated with an antibiotic. Second, pharmacoepidemiological studies, some of which minimized the protopathic bias, all converged and confirmed the risk. Third, experimental in vitro and in vivo animal studies suggest several biological mechanisms, which strengthens a causal link beyond the well-known risk of delaying the care of the infection (immunomodulatory effects, effects on S. pyogenes infections, and reduced antibiotics efficacy). Therefore, in case of infection, symptomatic treatment with NSAIDs for non-severe symptoms (fever, pain, or myalgia) is not to be recommended, given a range of clinical and scientific arguments supporting an increased risk of severe bacterial complication. Besides, the existence of a safer drug alternative, with paracetamol at recommended doses, makes this recommendation of precaution and common sense even more legitimate. In 2020, such recommendation is more topical than ever with the emergence of COVID-19, especially since it results in fever, headaches, muscular pain, and cough, and is further complicated with pneumopathy, and given experimental data suggesting a link between ibuprofen and the level of expression of angiotensin-converting enzyme 2.

Identification of Angiotensin I-Converting Enzyme-Inhibitory and Anticoagulant Peptides from Enzymatic Hydrolysates of Chicken Combs and Wattles.

Peptides from protein hydrolysate of a mixture of chicken combs and wattles (CCWs) were obtained through enzymatic hydrolysis, and their anticoagulant and inhibitory effects on angiotensin I-converting enzyme (ACE) were investigated. The protein hydrolysate exhibited anticoagulant capacity by the intrinsic pathway (activated partial thromboplastin time) and potent ACE-inhibitory activity. The peptides were sequenced by LC-MS to identify those with higher inhibitory potential. From the pool of sequenced peptides, the following three peptides were selected and synthesized based on their low molecular weight and the presence of amino acids with ACE-inhibitory potential at the C-terminus: peptide I (APGLPGPR), peptide II (Piro-GPPGPT), and peptide III (FPGPPGP). Peptide III (FPGPPGP) showed the highest ACE-inhibitory capacity among the peptides selected. In conclusion, a peptide (FPGPPGP) of unknown sequence was identified as having potent ACE-inhibitory capacity. This peptide originated from unconventional hydrolysates from poultry slaughter waste, including combs and wattles.

Identification of angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory peptides derived from oilseed proteins using two integrated bioinformatic approaches.

Angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) play critical roles in the development of hypertension and type 2 diabetes, respectively. Inhibiting ACE and DPP-IV activity using peptides has become part of new therapeutic strategies for supporting medicinal treatment of both diseases. In this study, oilseed proteins, including soybean, flaxseed, rapeseed, sunflower and sesame are evaluated for the possibility of generating ACE and DPP-IV inhibitory peptides using different integrated bioinformatic approaches (UniProt knowledgebase, ProtParam, BLAST, BIOPEP, PeptideRanker, Pepsite2 and ToxinPred), and three bovine proteins (ß-lactoglobulin, ß-casein and κ-casein) as comparisons. Compared with bovine proteins, the potency indices of ACE and DPP-IV inhibitory peptides, calculated using the BIOPEP database, suggest that oilseed proteins may be considered as good precursors of ACE inhibitory peptides but generate a relative lower yield of DPP-IV inhibitory peptides following subtilisin, pepsin (pH = 1.3) or pepsin (pH > 2) hydrolysis. Average scores aligned using PeptideRanker confirmed oilseed proteins as significant potential sources of bioactive peptides: over 105 peptides scored over 0.8. Pepsite2 predicted that these peptides would largely bind via Gln281, His353, Lys511, His513, Tyr520 and Tyr523 of ACE to inhibit the enzyme, while Trp629 would be the predominant binding site of peptides in reducing DPP-IV activity. All peptides were capable of inhibiting ACE and DPP-IV whilst 65 of these 105 peptides are not currently recorded in BIOPEP database. In conclusion, our in silico study demonstrates that oilseed proteins could be considered as good precursors of ACE and DPP-IV inhibitory peptides as well as so far unexplored peptides that potentially have roles in ACE and DPP-IV inhibition and beyond.

Forced degradation of l-(+)-bornesitol, a bioactive marker of Hancornia speciosa: Development and validation of stability indicating UHPLC-MS method and effect of degraded products on ACE inhibition.

The antihypertensive activity of the medicinal plant Hancornia speciosa has been previously demonstrated by us, being the activity ascribed to polyphenols and cyclitols like l-(+)-bornesitol. We herein evaluated the stability of the bioactive marker bornesitol submitted to forced degradation conditions. Bornesitol employed in the study was isolated from H. speciosa leaves. An UHPLC-ESI-MS/MS method was developed to investigate bornesitol stability based on MRM (Multiple Reaction Monitoring) acquisition mode and negative ionization mode, employing both specific (m/z 193 → 161 Da) and confirmatory (m/z 193 → 175 Da) transitions. A gradient elution of 0.1% formic acid in water and acetonitrile was performed on a HILIC column. The method was validated and showed adequate linearity (r2 > 0.99), selectivity, specificity, accuracy, and precision (RSD < 2.9%). The method was robust for deliberate variations on dessolvation temperature, but not for changes in the flow rate and dessolvation gas. The results from the stability studies allowed us to classify bornesitol as labile for acidic and alkaline hydrolysis, but as very stable for oxidative and neutral hydrolysis exposure. Bornesitol was categorized as practically stable under photolysis degradation, whereas a considerable reduction on its contents was induced by metal ions and thermolysis exposure. Degraded samples from neutral hydrolysis and thermolysis were assayed in vitro for ACE inhibition and showed a substantial decrease in biological activity as compared to intact bornesitol. myo-Inositol was identified as the major degradation products in both matrices. This is the first report on bornesitol stability under different stress conditions and the obtained data are relevant for the development and quality control of standardized products from H. speciosa leaves.

Purification of angiotensin-converting enzyme from human plasma and investigation of the effect of some active ingredients isolated from Nigella sativa L. extract on the enzyme activity.

In the present study, one-step purification of angiotensin-converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1), responsible for regulation of blood pressure, was achieved using affinity chromatography from human plasma. The enzyme was purified 12,860-fold with a specific activtiy of 5080 EU/mg protein. Optimum temperature and pH were determined for the enzyme as 35-40°C and pH 7.4-7.5, respectively. The purity of ACE was determined by SDS-PAGE and the enzyme showed two bands at 60 and 70 kDa on the gel. The native molecular weight of ACE was found to be 260 kDa by gel filtration chromatography, demonstrating that the enzyme has a heterodimeric structure. Natural fatty acids of Nigella sativa (Ranunculaceae) were isolated by means of column chromatography. The structures of these compounds were determined using NMR and GC-MS. The results showed that high concentrations of linoleic, oleic and palmitic acids were isolated from the plant. The effect of six fractions (Fr 1-6) on ACE activity was examined. Fraction 3 increased the ACE activity while the other fractions decreased the enzyme activity. The concentrations of the fractions inhibiting the half-maximum activity of the enzyme were calculated as 1.597 mg/mL for Fr 1, 0.053 mg/mL for Fr 2, 0.527 mg/mL for Fr 4, 0.044 mg/mL for Fr 5 and 0.136 mg/mL for Fr 6 using a Lineweaver-Burk graph.

Effects of Root Extracts of Eurycoma longifolia Jack on Corpus Cavernosum of Rat.

OBJECTIVE: This study was conducted to investigate the mechanisms of action of Eurycoma longifolia in rat corpus cavernosum. MATERIALS AND METHODS: Tincture of the roots was concentrated to dryness by evaporating the ethanol in vacuo. This ethanolic extract was partitioned into 5 fractions sequentially with hexane, dichloromethane (DCM), ethyl acetate, butanol, and water. The corpus cavernosum relaxant activity of each fraction was investigated. The DCM fraction which showed the highest potency in relaxing phenylephrine-precontracted corpora cavernosa was purified by column chromatography. The effects of the most potent DCM subfraction in relaxing phenylephrine-precontracted corpora cavernosa, DCM-I, on angiotensin I- or angiotensin II-induced contractions in corpora cavernosa were investigated. The effects of DCM-I pretreatment on the responses of phenylephrine-precontracted corpora cavernosa to angiotensin II or bradykinin were also studied. An in vitro assay was conducted to evaluate the effect of DCM-I on angiotensin-converting enzyme activity. RESULTS: Fraction DCM-I decreased the maximal contractions (100%) evoked by angiotensin I and angiotensin II to 30 ± 14% and 26 ± 16% (p < 0.001), respectively. In phenylephrine-precontracted corpora cavernosa, DCM-I pretreatment caused angiotensin II to induce 82 ± 27% relaxation of maximal contraction (p < 0.01) and enhanced (p < 0.001) bradykinin-induced relaxations from 47 ± 8% to 100 ± 5%. In vitro, DCM-I was able to reduce (p < 0.001) the maximal angiotensin-converting enzyme activity to 78 ± 0.24%. CONCLUSION: Fraction DCM-I was able to antagonize angiotensin II-induced contraction to cause corpus cavernosum relaxation via inhibition of angiotensin II type 1 receptor and enhance bradykinin-induced relaxation through inhibition of angiotensin-converting enzyme.

Comparative Effects of Alkaloid Extracts from Aframomum melegueta (Alligator Pepper) and Aframomum danielli (Bastered Melegueta) on Enzymes Relevant to Erectile Dysfunction.

Aframomum melegueta (alligator pepper (AP)) and Aframomum danielli (bastered melegueta (BM)) seeds have been known to improve sexual function in folkloric medicine. This study investigates the effects of AP and BM seeds' alkaloid extracts on the activities of enzymes (acetylcholinesterase (AChE), angiotensin-1-converting enzyme (ACE), phosphodiesterase-5 (PDE-5), and arginase) relevant to erectile dysfunction (ED). Alkaloids from the seeds were prepared by the solvent extraction method and their interactions with AChE, ACE, PDE-5, and arginase were assessed. Gas chromatographic (GC) analyses of the extracts were also performed. The results revealed that the extracts inhibited the enzymes in a concentration-dependent manner. However, alkaloid extract from AP seed had higher AChE (IC50 = 5.42 µg/mL) and ACE (IC50 = 12.57 µg/mL) but lower PDE-5 (IC50 = 33.80 µg/mL) and arginase (IC50 = 31.36 µg/mL) inhibitory effects when compared to that of BM extract (AChE, IC50 = 42.00; ACE, IC50 = 60.67, PDE-5, IC50 = 7.24; and arginase, IC50 = 2.53 µg/mL). The GC analyses revealed the presence of senkirkine, angustifoline, undulatine, myristicin, safrole, lupanine, powelle, and indicine-N-oxide, among others. The inhibition of these enzymes could be the possible mechanisms by which the studied seeds were being used in managing ED in folklores. Nevertheless, the seed of AP exhibited higher potentials.

Dietary peptides from the non-digestible fraction of Phaseolus vulgaris L. decrease angiotensin II-dependent proliferation in HCT116 human colorectal cancer cells through the blockade of the renin-angiotensin system.

This study aimed to determine the ability of peptides present in the non-digestible fraction (NDF) of common beans to decrease angiotensin II (AngII) through the blockade of RAS and its effect on the proliferation of HCT116 human colorectal cancer cells. Pure synthesized peptides GLTSK and GEGSGA and the peptide fractions (PF) of cultivars Azufrado Higuera and Bayo Madero were used. The cells were pretreated with pure peptides, PF or AGT at their IC50 or IC25 values, in comparison with the simultaneous treatment of peptides and AGT. For western blot and microscopy analysis, 100 µM and 0.5 mg mL(-1) were used for pure peptides and PF treatments, respectively. According to the ELISA tests, GLTSK and GEGSGA decreased (p < 0.05) the conversion rate of AGT to angiotensin I (AngI) by 38 and 28%, respectively. All the peptides tested reduced (p < 0.05) the conversion rate of AngI to AngII from 38 to 50%. When the cells were pretreated with both pure peptides and PF before exposure to AGT, the effectiveness inhibiting cell proliferation was higher than the simultaneous treatment suggesting their preventive effects. GLTSK and GEGSGA interacted with the catalytic site of renin, the angiotensin-I converting enzyme, and the AngII receptor, mainly through hydrogen bonds, polar, hydrophobic and cation-π interactions according to molecular docking. Through confocal microscopy, it was determined that GLTSK and GEGSGA caused the decrease (p < 0.05) of AngII-dependent STAT3 nuclear activation in HCT116 cells by 66 and 23%, respectively. The results suggest that peptides present in the common bean NDF could potentially ameliorate the effects of RAS overexpression in colorectal cancer.

Ocular Inserts for Sustained Release of the Angiotensin-Converting Enzyme 2 Activator, Diminazene Aceturate, to Treat Glaucoma in Rats.

The aim of this study was to develop and evaluate the effects of chitosan inserts for sustained release of the angiotensin-converting enzyme 2 (ACE2) activator, diminazene aceturate (DIZE), in experimental glaucoma. Monolayer DIZE loaded inserts (D+I) were prepared and characterized through swelling, attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and in vitro drug release. Functionally, the effects of D+I were tested in glaucomatous rats. Glaucoma was induced by weekly injections of hyaluronic acid (HA) into the anterior chamber and intraocular pressure (IOP) measurements were performed. Retinal ganglion cells (RGC) and optic nerve head cupping were evaluated in histological sections. Biodistribution of the drug was accessed by scintigraphic images and ex vivo radiation counting. We found that DIZE increased the swelling index of the inserts. Also, it was molecularly dispersed and interspersed in the polymeric matrix as a freebase. DIZE did not lose its chemical integrity and activity when loaded in the inserts. The functional evaluation demonstrated that D+I decreased the IOP and maintained the IOP lowered for up to one month (last week: 11.0 ± 0.7 mmHg). This effect of D+I prevented the loss of RGC and degeneration of the optic nerve. No toxic effects in the eyes related to application of the inserts were observed. Moreover, biodistribution studies showed that D+I prolonged the retention of DIZE in the corneal site. We concluded that D+I provided sustained DIZE delivery in vivo, thereby evidencing the potential application of polymeric-based DIZE inserts for glaucoma management.

A novel ACE2 activator reduces monocrotaline-induced pulmonary hypertension by suppressing the JAK/STAT and TGF-ß cascades with restored caveolin-1 expression.

INTRODUCTION: Pulmonary hypertension (PH) is characterized by increased pressure in the pulmonary artery and right ventricular hypertrophy (RVH). Recently, angiotensin-converting enzyme 2 (ACE2), which converts angiotensin (Ang) II into Ang-(1-7), was shown to inhibit experimental PH. Here we identified a novel ACE2 activator and investigated how the compound reduced monocrotaline (MCT)-induced PH. METHODS: To induce PH, Sprague-Dawley rats were injected subcutaneously with MCT, followed by the continuous administration of NCP-2454, an ACE2 activator, using osmotic pumps. Pulmonary arterial compliance was monitored every week until 4 weeks post-injection (wpi). RVH and lung remodeling was evaluated using lung tissue at 4 wpi. RESULTS: NCP-2454 upregulated the production of Ang-(1-7) when incubated with ACE2 and Ang II. Notably, a continuous infusion of NCP-2454 significantly improved pulmonary arterial compliance, right ventricular systolic pressure, and RVH in MCT-treated rats. Interestingly, NCP-2454 increased the relative expression of ACE2 and MAS mRNA in lung tissue, especially in MCT-treated rats. In addition, the compound inhibited the MCT-induced overexpression of transforming growth factor ß, phosphorylation of signal transducer and activator of transcription-3 (STAT3), and interleukin-6 production. The compound also restored the expression of caveolin-1 (Cav-1), which negatively regulates the Janus kinase-STAT signaling cascade. CONCLUSIONS: NCP-2454 prevented MCT-induced PH by suppressing intracellular inflammatory cascades, an upstream molecular change of which is the disruption of Cav-1 expression.

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

ETHNOPHARMACOLOGICAL RELEVANCE: Sandpaper [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 METHODS: The 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. RESULTS: The 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. CONCLUSION: The 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.