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Breast cancer (BRCA) is a prevalent malignancy with the highest incidence among females. BRCA can be categorized into five intrinsic molecular subtypes (LumA, LumB, HER2, Basal, and Normal), each characterized by varying molecular and clinical features determined by the expression of intrinsic genes (PAM50). The Heat Shock Protein (HSP) family is composed of 95 genes evolutionary conservated, they have critical roles in proteostasis in both normal and cancerous processes. Many studies have linked HSP to the development and spread of cancer. They modulate the activity of multiple proteins expressed by oncogenes and anti-oncogenes through a range of interactions. In this study, we evaluate the mutational changes that HSP undergoes in BRCA mainly from the TCGA database. We observe that Copy Number Variations (CNV) are the more frequent events analyzed surpassing the occurrence of point mutations, indels, and translation start site mutations. The Basal subtype showcased the highest count of amplified CNV, including subtype-specific changes, whereas the Luminals tumors accumulated the greatest number of deletion CNV. Meanwhile, the HER2 subtype exhibited a comparatively lower frequency of CNV alterations when compared to the other subtypes. This study integrates CNV and expression data, finding associations between these two variables and the influence of CNV on the deregulation of HSP expression. To enhance the role of HSP as a risk predictor in BRCA, we succeeded in identifying CNV profiles as a prognostic marker. We included Artificial Intelligence to improve the clustering of patients, and we achieved a molecular CNV signature as a significant risk factor independent of known classic markers, including molecular subtypes PAM50. This research enhances the comprehension of HSP DNA alterations in BRCA and its relation with predicting the risk of affected individuals providing insights to develop guide personalized treatment strategies.
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Neoplasias de la Mama , Variaciones en el Número de Copia de ADN , Proteínas de Choque Térmico , Mutación , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Proteínas de Choque Térmico/genética , Regulación Neoplásica de la Expresión Génica , Biomarcadores de Tumor/genéticaRESUMEN
Morin (MRN) is a natural compound with antiangiogenic, antioxidant, anti-inflammatory, and anticancer activity. However, it shows a very low water solubility (28 µg/mL) that reduces its oral absorption, making bioavailability low and unpredictable. To improve MRN solubility and positively affect its biological activity, particularly its antiangiogenic activity, in this work, we prepared the inclusion complexes of MNR with sulfobutylether-ß-cyclodextrin (SBE-ß-CD) and hydroxypropyl-ß-cyclodextrin (HP-ß-CD). The inclusion complexes obtained by the freeze-drying method were extensively characterized in solution (phase-solubility studies, UV-Vis titration, and NMR spectroscopy) and in the solid state (TGA, DSC, and WAXD analysis). The complexation significantly increased the water solubility by about 100 times for MRN/HP-ß-CD and 115 times for MRN/SBE-ß-CD. Furthermore, quantitative dissolution of the complexes was observed within 60 min, whilst 1% of the free drug dissolved in the same experimental time. 1H NMR and UV-Vis titration studies demonstrated both CDs well include the benzoyl moiety of the drug. Additionally, SBE-ß-CD could interact with the cinnamoyl moiety of MRN too. The complexes are stable in solution, showing a high value of association constant, that is, 3380 M-1 for MRN/HP-ß-CD and 2870 M-1 for MRN/SBE-ß-CD. In vivo biological studies on chick embryo chorioallantoic membrane (CAM) and zebrafish embryo models demonstrated the high biocompatibility of the inclusion complexes and the effective increase in antiangiogenic activity of complexed MRN with respect to the free drug.
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Tyrosinase (EC 1.14.18.1) is implicated in melanin production in various organisms. There is a growing body of evidence suggesting that the overproduction of melanin might be related to several skin pigmentation disorders as well as neurodegenerative processes in Parkinson's disease. Based on this consideration, the development of tyrosinase inhibitors represents a new challenge to identify new agents in pharmaceutical and cosmetic applications. With the goal of identifying tyrosinase inhibitors from a synthetic source, we employed a cheap and facile preliminary assay using tyrosinase from Agaricus bisporus (AbTYR). We have previously demonstrated that the 4-fluorobenzyl moiety might be effective in interactions with the catalytic site of AbTYR; moreover, the additional chlorine atom exerted beneficial effects in enhancing inhibitory activity. Therefore, we planned the synthesis of new small compounds in which we incorporated the 3-chloro-4-fluorophenyl fragment into distinct chemotypes that revealed the ability to establish profitable contact with the AbTYR catalytic site. Our results confirmed that the presence of this fragment is an important structural feature to improve the AbTYR inhibition in these new chemotypes as well. Furthermore, docking analysis supported the best activity of the selected studied compounds, possessing higher potency when compared with reference compounds.
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Agaricus , Monofenol Monooxigenasa , Monofenol Monooxigenasa/metabolismo , Melaninas/farmacología , Agaricus/química , Dominio Catalítico , Inhibidores Enzimáticos/química , Simulación del Acoplamiento MolecularRESUMEN
Leishmaniasis is a neglected disease caused by flagellated parasites of the Leishmania genus affecting more than 10 million people worldwide. Current treatments for leishmaniasis involve the administration of poorly tolerated drugs with toxic side effects in patients. There is an imperative necessity for novel compounds to treat this disease. One of the most used strategies in the search for different antiparasitic compounds is the screening of purified plant molecules. The diterpenes 12-hydroxy-11,14-diketo-6,8,12-abietatrien-19,20-olide (HABTO) and 5-epi-icetexone (ICTX) isolated from Salvia cuspidata were shown to be effective against Leishmania amazonensis in vitro and in vivo. They displayed an antiproliferative effect against L. amazonensis promastigotes. They also induce an increase in ROS levels and affect the mitochondrial activity of parasites. HABTO and ICTX in an in vivo model of cutaneous leishmaniasis decrease footpad swelling, parasite load, and splenic index. Moreover, they induce significant reduction in the O.D. of total anti-Leishmania IgG and IgG1 subtype antibody responses against L. amazonensis compared to the PBS group but maintain high levels of IgG2a. This suggests that in HABTO- or ICTX-treated mice, there is a slowdown in the progression of the disease. These terpenes could be considered as possible novel antileishmanial agents against L. amazonensis and thus treat cutaneous leishmaniasis.
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Antiprotozoarios , Leishmania mexicana , Leishmania , Leishmaniasis Cutánea , Salvia , Animales , Ratones , Antiparasitarios/farmacología , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Leishmaniasis Cutánea/tratamiento farmacológico , Ratones Endogámicos BALB C , Terpenos/farmacologíaRESUMEN
Novel adjuvants are highly desired to improve immune responses of SARS-CoV-2 vaccines. This work reports the potential of the stimulator of interferon genes (STING) agonist adjuvant, the cyclic di-adenosine monophosphate (c-di-AMP), in a SARS-CoV-2 vaccine based on the receptor binding domain (RBD). Here, mice immunized with two doses of monomeric RBD adjuvanted with c-di-AMP intramuscularly were found to exhibit stronger immune responses compared to mice vaccinated with RBD adjuvanted with aluminum hydroxide (Al(OH)3 ) or without adjuvant. After two immunizations, consistent enhancements in the magnitude of RBD-specific immunoglobulin G (IgG) antibody response were observed by RBD + c-di-AMP (mean: 15360) compared to RBD + Al(OH)3 (mean: 3280) and RBD alone (n.d.). Analysis of IgG subtypes indicated a predominantly Th1-biased immune response (IgG2c, mean: 14480; IgG2b, mean: 1040, IgG1, mean: 470) in mice vaccinated with RBD + c-di-AMP compared to a Th2-biased response in those vaccinated with RBD + Al(OH)3 (IgG2c, mean: 60; IgG2b: n.d.; IgG1, mean: 16660). In addition, the RBD + c-di-AMP group showed better neutralizing antibody responses as determined by pseudovirus neutralization assay and by plaque reduction neutralization assay with SARS-CoV-2 wild type. Moreover, the RBD + c-di-AMP vaccine promoted interferon-γ secretion of spleen cell cultures after RBD stimulation. Furthermore, evaluation of IgG-antibody titers in aged mice showed that di-AMP was able to improve RBD-immunogenicity at old age after 3 doses (mean: 4000). These data suggest that c-di-AMP improves immune responses of a SARS-CoV-2 vaccine based on RBD, and would be considered a promising option for future COVID-19 vaccines.
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Vacunas contra la COVID-19 , COVID-19 , Animales , Ratones , Humanos , SARS-CoV-2 , Adyuvantes Inmunológicos , Inmunidad Celular , Anticuerpos Neutralizantes , Adyuvantes Farmacéuticos , Inmunoglobulina G , Adenosina Monofosfato , Anticuerpos Antivirales , Glicoproteína de la Espiga del Coronavirus , Inmunidad HumoralRESUMEN
In brief: The endocrine and immunological disruption induced by hyperthyroidism could alter gestation, placenta, and fetal development. This study suggests an immunological role of thyroid hormones in gestation. Abstract: Thyroid dysfunctions lead to metabolic, angiogenic, and developmental alterations at the maternal-fetal interface that cause reproductive complications. Thyroid hormones (THs) act through their nuclear receptors that interact with other steroid hormone receptors. Currently, immunological regulation by thyroid status has been characterized to a far less extent. It is well known that THs exert regulatory function on immune cells and modulate cytokine expression, but how hyperthyroidism (hyper) modulates placental immunological aspects leading to placental alterations is unknown. This work aims to throw light on how hyper modulates immunological and morphological placental aspects. Control and hyper (induced by a daily s.c. injection of T4 0.25 mg/kg) Wistar rats were mated 8 days after starting T4 treatment and euthanized on days 19 (G19) and 20 (G20) of pregnancy. We removed the placenta to perform qPCR, flow cytometry, immunohistochemistry, Western blot and histological analysis, and amniotic fluid and serum to evaluate hormone levels. We observed that hyper increases the fetal number, fetal weight, and placental weight on G19. Moreover, hyper induced an endocrine imbalance with higher serum corticosterone and changed placental morphology, specifically the basal zone and decidua. These changes were accompanied by an increased mRNA expression of glucocorticoid receptor and monocyte chemoattractant protein-1, an increased mRNA and protein expression of prolactin receptor, and an increase in CD45+ infiltration. Finally, by in vitro assays, we evidenced that TH induced immune cell activation. In summary, we demonstrated that hyper modulates immunological and morphological placental aspects and induces fetal phenotypic changes, which could be related to preterm labor observed in hyper.
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Hipertiroidismo , Placenta , Ratas , Animales , Embarazo , Femenino , Placenta/metabolismo , Ratas Wistar , Hormonas Tiroideas/metabolismo , Hipertiroidismo/metabolismo , Hipertiroidismo/patología , ARN Mensajero/metabolismo , Leucocitos/metabolismoRESUMEN
In the present study, we focused our attention on Crataegus laciniata Ucria (Rosaceae), which is wild growing in western Sicily (Italy). The chemical profile of the C. laciniata flower's (CLF) ethanolic (70%) extract showed the presence of both C-flavonoid and O-flavonoid derivatives. Beyond the main metabolites, like hyperoside and vitexin, there are several luteolin derivates, in addition to catechin and epicatechin dimers or trimers. Regarding the antioxidant activities, CLF showed a strong ability to scavenge DPPH and ABTS radicals and a good Fe3+-reducing antioxidant power. The investigation into the key enzymes in diabetes showed strong inhibition on α-amylase and α-glucosidase, whereas the skin-whitening properties are linked to inhibitory effects on tyrosinase. Moreover, we employed Danio rerio (zebrafish) for toxicity assessment, as it represents an ideal in vivo model due to its high correlation with humans in response to pharmaceutical and cosmetic testing. Zebrafish embryos exposed to CLF (25-100 µg/mL) showed marked depigmentation compared to phenylthiourea (PTU), in addition to a high survival percentage and the absence of malformations. In conclusion, this experimental study outlines that C. laciniata flowers could be a potential source of bioactive compounds for application in the pharmaceutical and cosmeceutical industries.
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Monarda didyma L. (Lamiaceae) is a medicinal and aromatic herb native to eastern North America and now is also cultivated in Northern Italy, which shows terminal heads of bright scarlet-red flowers, subtended by a whorl of red-tinged leafy bracts. Starting from 2018, M. didyma flowering tops have been included in the Belfrit List of botanicals. However, to date studies on the crude extract of this plant are still lacking. The aim of the present study was to investigate the morphological and anatomical features of the flowering tops and the phytochemical profile of their ethanolic and hydroglyceric extracts (EE and HGE, respectively). HGE was the richest in total phenols (105.75 ± 5.91 vs. 64.22 ± 3.45 mg/100 mL) and especially in flavonoids (71.60 ± 5.09 vs. 47.70 ± 1.27 mg/100 mL), as confirmed also by LC-DAD-ESI-MS. Fifty-three polyphenols were identified and quantified. Even if they showed a common polyphenolic profile, EE and HGE showed quantitative differences. Flavan-3-ols and anthocyanins were the most expressed metabolites in HGE, whereas flavonols were the most expressed metabolites in EE. These features confer to HGE the highest antioxidant, anti-inflammatory, and anti-angiogenic properties, detected by several in vitro and in vivo assays, highlighting a promising use of this plant extract for skincare applications.
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Transition metal complexes have continued to constitute an appealing class of medicinal compounds since the exceptional discovery of cisplatin in the late 1960s. Pt(II)-based complexes are endowed with a broad range of biological properties, which are mainly exerted by targeting DNA. In this study, we report a significant biological investigation into and computation analyses of four Pt(II)-complexes, namely, LDP-1-4, synthesized and characterized according to previously reported procedures. Molecular-modelling studies highlighted that the top two LDP compounds (i.e., LDP-1 and LDP-4) might bind to both matched and mismatched base pair sites of the oligonucleotide 5'-(dCGGAAATTACCG)2-3', supporting their anticancer potential. These two complexes displayed noteworthy cytotoxicity in vitro (sub-micromolar-micromolar range) against two leukaemia cell lines, i.e., CCRF-CEM and its multi-drug-resistant counterpart CEM/ADR5000, and remarkable anti-angiogenic properties (in the sub-micromolar range) evaluated in an in vivo model, i.e., a chick embryo chorioallantoic membrane (CAM) assay.
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Antineoplásicos , Complejos de Coordinación , Animales , Embrión de Pollo , Platino (Metal)/farmacología , Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Cisplatino , ADN , Línea Celular TumoralRESUMEN
Alnus glutinosa (namely black alder or European alder) is a tree of the Betulaceae family widely spread through Europe, Southeastern Asia, the Caucasus mountains, and Western Siberia. Its bark is traditionally used for medicinal purposes as an astringent, cathartic, febrifuge, emetic, hemostatic, and tonic, suggesting that it may contain bioactive compounds useful to counteract inflammation. The aim of this study was to investigate the phytochemical profile of A. glutinosa stem bark extract (AGE) by LC-DAD-ESI-MS/MS analysis and to validate some biological activities such as antioxidant, anti-inflammatory and anti-angiogenic properties by in vitro and in vivo models (chick chorioallantoic membranes and zebrafish embryos), that can justify its use against inflammatory-based diseases. The AGE showed a high total phenols content expressed as gallic acid equivalents (0.71 g GAE/g of AGE). Diarylheptanoids have been identified as the predominant compounds (0.65 g/g of AGE) with oregonin, which alone constitutes 74.67% of the AGE. The AGE showed a strong and concentration-dependent antioxidant (IC50 0.15-12.21 µg/mL) and anti-inflammatory (IC50 5.47-12.97 µg/mL) activity. Furthermore, it showed promising anti-angiogenic activity, inhibiting both the vessel growth (IC50 23.39 µg/egg) and the release of an endogenous phosphatase alkaline enzyme (IC50 44.24 µg/embryo). In conclusion, AGE is a promising source of antioxidant, anti-inflammatory and angio-modulator compounds.
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Melanin biosynthesis is enzymatically regulated by tyrosinase (TYR, EC 1.14.18.1), which is efficiently inhibited by natural and synthetic phenols, demonstrating potential therapeutic application for the treatment of several human diseases. Herein we report the inhibitory effects of a series of (4-(4-hydroxyphenyl)piperazin-1-yl)arylmethanone derivatives, that were designed, synthesised and assayed against TYR from Agaricus bisporus (AbTYR). The best inhibitory activity was predominantly found for compounds bearing selected hydrophobic ortho-substituents on the aroyl moiety (IC50 values in the range of 1.5-4.6â µM). They proved to be more potent than the reference compound kojic acid (IC50 =17.8â µM) and displayed competitive mechanism of inhibition of diphenolase activity of AbTYR. Docking simulation predicted their binding mode into the catalytic cavities of AbTYR and the modelled human TYR. In addition, these compounds displayed antioxidant activity combined with no cytotoxicity in MTT tests. Notably, the best inhibitor affected tyrosinase activity in α-MSH-stimulated B16F10 cells, thus demonstrating anti-melanogenic activity.
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Inhibidores Enzimáticos , Monofenol Monooxigenasa , Humanos , Piperazina/farmacología , Relación Estructura-Actividad , Inhibidores Enzimáticos/química , Estructura Molecular , Relación Dosis-Respuesta a Droga , Simulación del Acoplamiento MolecularRESUMEN
Background and aim: Prosopis strombulifera (Lam.) Benth is a rhizomatous shrub native from different zones of Argentine Republic. P. strombulifera aqueous extract (PsAE) has different effects and several biological activities have been reported. The goal of this study was to analyze the activity of PsAE on a murine model of cutaneous leishmaniasis caused by Leishmania amazonensis. Experimental procedure: PsAE was orally administered at 150 mg/animal/day on BALB/c mice infected in the right footpad (RFP) with 1 × 105 promastigotes of L. amazonensis. As a chemotherapeutic control of treatment, animals receive a commercial form of meglumine antimoniate (MA) (Glucantime®, Aventis, Paris, France). Results and conclusion: We observe that the size of RFP lesions of infected mice without treatment showed a grade of inflammation, ulceration and necrosis at the site of infection much greater than that observed with PsAE or MA treatment. Moreover, PsAE was capable of decreasing parasite burden and splenic index. Furthermore, PsAE treated mice showed a significant decrease in O.D. of total anti-Leishmania IgG antibody responses against L. amazonensis. This decrease was similar to those observed when the reference drug, MA, was used. This would indicate that PsAE treatment inhibits or delays disease progression in mice. In conclusion, our findings suggest that PsAE could be a potential candidate to be used, as a new therapeutic strategy, to treat cutaneous leishmaniasis caused by L. amazonensis.
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The methanolic extract of the stem bark of a wild species of jujube, Ziziphus lotus (L.) Lam., growing in Sicily, was chemically and biologically investigated. The chemical profile was defined by UHPLC-HR-ESI-Orbitrap/MS analysis whereas antioxidant and tyrosinase inhibitory activities were investigated by inâ vitro assays. The extract showed a high total phenolic and flavonoid content (TPC=271.65â GAE/g and TFC=188.11â RE/g extract). Metabolomic analysis revealed a rich phytocomplex characterized by phenols, cyclopeptide alkaloids, and triterpenoid saponins, some of which here detected for the first time. The mushroom tyrosinase inhibition assay displayed that the methanolic extract efficiently inhibits the monophenolase and diphenolase activity. Furthermore, the extract showed a strong ability to scavenge DPPH, a good Fe3+ reducing antioxidant power, in addition to a Fe2+ chelating activity. Taken together, these results suggest possible novel applications of wild jujube stem bark as a source of potential skin-care agents with several uses in pharmaceutical and cosmetic industries.
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Promoción de la Salud , Ziziphus , Antioxidantes/química , Flavonoides/farmacología , Monofenol Monooxigenasa , Fenoles/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Ziziphus/químicaRESUMEN
Leishmaniasis is a neglected tropical disease (NTD) caused by parasites belonging to the Leishmania genus for which there is no vaccine available for human use. Thus, the aims of this study are to evaluate the immunoprotective effect of a first-generation vaccine against L. amazonensis and to identify its immunodominant antigens. BALB/c mice were inoculated with phosphate buffer sodium (PBS), total L. amazonensis antigens (TLAs), or TLA with Poly (I:C) and Montanide ISA 763. The humoral and cellular immune response was evaluated before infection. IgG, IgG1, and IgG2a were measured on serum, and IFN-γ, IL-4, and IL-10 cytokines as well as cell proliferation were measured on a splenocyte culture from vaccinated mice. Immunized mice were challenged with 104 infective parasites of L. amazonensis on the footpad. After infection, the protection provided by the vaccine was analyzed by measuring lesion size, splenic index, and parasite load on the footpad and spleen. To identify immunodominant antigens, total proteins of L. amazonensis were separated on 2D electrophoresis gel and transferred to a membrane that was incubated with serum from immunoprotected mice. The antigens recognized by the serum were analyzed through a mass spectrometric assay (LC-MS/MS-IT-TOF) to identify their protein sequence, which was subjected to bioinformatic analysis. The first-generation vaccine induced higher levels of antibodies, cytokines, and cell proliferation than the controls after the second dose. Mice vaccinated with TLA + Poly (I:C) + Montanide ISA 763 showed less footpad swelling, a lower splenic index, and a lower parasite load than the control groups (PBS and TLA). Four immunodominant proteins were identified by mass spectrometry: cytosolic tryparedoxin peroxidase, an uncharacterized protein, a kinetoplast-associated protein-like protein, and a putative heat-shock protein DNAJ. The identified proteins showed high levels of conserved sequence among species belonging to the Leishmania genus and the Trypanosomatidae family. These proteins also proved to be phylogenetically divergent to human and canine proteins. TLA + Poly (I:C) + Montanide ISA 763 could be used as a first-generation vaccine against leishmaniasis. The four proteins identified from the whole-protein vaccine could be good antigen candidates to develop a new-generation vaccine against leishmaniasis.
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Leishmania , Leishmaniasis Cutánea , Vacunas , Animales , Cromatografía Liquida , Citocinas/metabolismo , Perros , Epítopos Inmunodominantes , Leishmaniasis Cutánea/prevención & control , Ratones , Aceite Mineral , Poli I-C , Espectrometría de Masas en TándemRESUMEN
A novel series of twenty-seven cinnamides constituted by cinnamic acid derivatives liked to 1-aryl piperazines were synthesized and evaluated for their potential inhibitory diphenolase activity of mushroom tyrosinase. Among them, the presence of a 3-chloro-4-fluorophenyl moiety at the N-1 position of piperazine ring was essential for a potent tyrosinase inhibitory effect, with the 3-nitrocinnamoyl (19p) and 2-chloro-3-methoxycinnamoyl (19t) derivatives as the most potent compounds of the series, with IC50 of 0.16 and 0.12 µM, respectively, resulting much active than kojic acid, whose IC50 value was 17.76 µM. In general, all compounds characterized by the presence of a 1-(3-chloro-4-fluorophenyl)piperazine moiety showed an excellent potency, and the nature, position and number of the substituents on the aryl of the cinnamic acid did not affect significantly the anti-tyrosinase activity. The molecular docking to the active site of the enzyme has been also performed to investigate the nature of enzyme-inhibitor interactions. Furthermore, for selected highly active compounds, their ability to inhibit melanogenesis in the A375 human melanoma cells and in vivo zebrafish model was also evaluated. One of the most potent compounds of series (19t) significantly reduced the pigmentation of zebrafish at 50 µM, unfortunately showing 100% mortality in the Fish Embryo Acute Toxicity (FET) test at the same concentration, Moreover, the zebrafish assay reveals that also compound 19r (IC50:0.51 µM against mushroom tyrosinase) effectively reduces melanogenesis with no acute toxicity effects and it could be proposed as potential candidate to treat tyrosinase-mediated hyperpigmentation.
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Agaricales , Monofenol Monooxigenasa , Animales , Cinamatos , Inhibidores Enzimáticos/química , Humanos , Melaninas , Simulación del Acoplamiento Molecular , Pez CebraRESUMEN
In this study, Mentha pulegium leaves and flowers harvested in three different Sicilian areas were investigated from a micromorphological, phytochemical and biological point of view. Light and scanning electron microscopy showed the presence of spherocrystalline masses of diosmin both in the leaf epidermal cells and in thin flower petals. Two different chemotypes were identified (I, kaempferide/rosmarinic acid; II, jaceidin isomer A). Phytochemical screening identified plant from collection site II as the richest in total phenolics (16.74 g GAE/100 g DE) and that from collection site I as the richest in flavonoids (46.56 g RE/100 g DE). Seventy-seven metabolites were identified both in flower and leaf extracts. Plant from site II showed the best antioxidant (0.90-83.72 µg/mL) and anti-inflammatory (27.44-196.31 µg/mL) activity expressed as half-maximal inhibitory concentration (IC50) evaluated by DPPH, TEAC, FRAP, ORAC, BSA denaturation and protease inhibition assays. These data were also corroborated by in vitro cell-based assays on lymphocytes and erythrocytes. Moreover, plant of site II showed the best antiangiogenic properties (IC50 33.43-33.60 µg/mL) in vivo on a chick chorioallantoic membrane. In conclusion, pedoclimatic conditions influence the chemotype and the biological activity of M. pulegium, with chemotype I showing the most promising biological properties.
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Leishmaniasis is a neglected tropical disease (NTD) caused by parasites belonging to the Leishmania genus for which there is no vaccine available for human use. Thus, the aims of this study are to evaluate the immunoprotective effect of a first-generation vaccine against L. amazonensis and to identify its immunodominant antigens. BALB/c mice were inoculated with phosphate buffer sodium (PBS), total L. amazonensis antigens (TLAs), or TLA with Poly (I:C) and Montanide ISA 763. The humoral and cellular immune response was evaluated before infection. IgG, IgG1, and IgG2a were measured on serum, and IFN-γ, IL-4, and IL-10 cytokines as well as cell proliferation were measured on a splenocyte culture from vaccinated mice. Immunized mice were challenged with 104 infective parasites of L. amazonensis on the footpad. After infection, the protection provided by the vaccine was analyzed by measuring lesion size, splenic index, and parasite load on the footpad and spleen. To identify immunodominant antigens, total proteins of L. amazonensis were separated on 2D electrophoresis gel and transferred to a membrane that was incubated with serum from immunoprotected mice. The antigens recognized by the serum were analyzed through a mass spectrometric assay (LC-MS/MS-IT-TOF) to identify their protein sequence, which was subjected to bioinformatic analysis. The first-generation vaccine induced higher levels of antibodies, cytokines, and cell proliferation than the controls after the second dose. Mice vaccinated with TLA + Poly (I:C) + Montanide ISA 763 showed less footpad swelling, a lower splenic index, and a lower parasite load than the control groups (PBS and TLA). Four immunodominant proteins were identified by mass spectrometry: cytosolic tryparedoxin peroxidase, an uncharacterized protein, a kinetoplast-associated protein-like protein, and a putative heat-shock protein DNAJ. The identified proteins showed high levels of conserved sequence among species belonging to the Leishmania genus and the Trypanosomatidae family. These proteins also proved to be phylogenetically divergent to human and canine proteins. TLA + Poly (I:C) + Montanide ISA 763 could be used as a first-generation vaccine against leishmaniasis. The four proteins identified from the whole-protein vaccine could be good antigen candidates to develop a new-generation vaccine against leishmaniasis.
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ABSTRACT Leishmaniasis is a group of parasitic zoonotic diseases caused by intracellular protozoans belonging to the genusLeishmania. Little is known about the effects that this parasitosis may have on the reproductive parameters and pregnancy of infected humans and pets. This study aimed to evaluate the influence of chronic cutaneous leishmaniasis caused byLeishmania (Leishmania) amazonensison reproductive and fetal parameters using a female murine model. A control group of female BALB/c mice and a group infected withL. (L.) amazonensiswere mated with healthy males. Clinical parameters were monitored during the pre-mating and gestational periods. Female mice were euthanized on day 19 of gestation, when the fetuses were weighed and their length measured and embryonic resorptions and fetal death were recorded. We observed five fetal deaths and three embryonic resorptions in the infected group. Furthermore, there was a decrease in fertility in the infected group (26.32%). The weight of the offspring from infected mothers was lower than that in the control group (1.019±0.035g and 1.163±0.032g,p<0.01). Fetal length was reduced in the infected group (3.71±0.05cm in the control group and 3.40±0.06cm in the infected groupp<0.001). This study shows that cutaneous leishmaniasis caused byL. (L.) amazonensisimpairs reproductive and fetal parameters in mice.
RESUMEN La leishmaniasis comprende un grupo de enfermedades zoonóticas parasitarias causadas por protozoos intracelulares pertenecientes al géneroLeishmania. Poco se conoce sobre los efectos que esta parasitosis puede tener sobre los parámetros reproductivos y la gestación en humanos y en otras especies infectadas. El objetivo de este estudio fue determinar la influencia de la leishmaniasis cutánea crónica, causada porLeishmania (Leishmania) amazonensis, en parámetros reproductivos y fetales. Se apareó un grupo control y un grupo infectado de ratones hembra BALB/c (previamente inoculado conL. (L.) amazonensis) con machos sanos. Se analizaron parámetros clínicos durante los períodos pregestacional y gestacional. Las hembras fueron eutanasiadas en el día 19 de gestación, momento en el cual se pesaron y midieron los fetos y se registraron las reabsorciones embrionarias y las muertes fetales. Se observaron 5 muertes fetales y 3 reabsorciones embrionarias en el grupo infectado. Además, hubo una disminución en la fertilidad de este último grupo (26,32%). Por otra parte, el peso de la descendencia de madres infectadas fue menor que el del grupo control (1,019±0,035g y 1,163±0,032g, respectivamente,p<0,01). Por último, la longitud fetal se redujo en el grupo infectado (3,71±0,05cm en el grupo control y 3,40±0,06cm en el grupo infectado,p<0,001). Este estudio muestra que la leishmaniasis cutánea causada porL. (L.) amazonensisafecta los parámetros reproductivos y fetales en ratones.
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Animales , Femenino , Masculino , Ratones , Embarazo , Leishmaniasis Cutánea , Leishmania , Feto , Ratones Endogámicos BALB CRESUMEN
There is a considerable attention for the development of inhibitors of tyrosinase (TYR) as therapeutic strategy for the treatment of hyperpigmentation disorders in humans. Continuing in our efforts to identify TYR inhibitors, we describe the design, synthesis and pharmacophore exploration of new small molecules structurally characterized by the presence of the 4-fluorobenzylpiperazine moiety as key pharmacophoric feature for the inhibition of TYR from Agaricus bisporus (AbTYR). Our investigations resulted in the discovery of the competitive inhibitor [4-(4-fluorobenzyl)piperazin-1-yl]-(3-chloro-2-nitro-phenyl)methanone 26 (IC50 =0.18â µM) that proved to be â¼100-fold more active than reference compound kojic acid (IC50 =17.76â µM). Notably, compound 26 exerted antimelanogenic effect on B16F10 cells in absence of cytotoxicity. Docking analysis suggested its binding mode into AbTYR and into modelled human TYR.
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Inhibidores Enzimáticos/farmacología , Piperazina/farmacología , Agaricus/enzimología , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Monofenol Monooxigenasa , Piperazina/síntesis química , Piperazina/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The genus Rhanterium (Asteraceae) is a widely distributed medicinal plant throughout western North Africa and some Rhanterium species are used in folk medicine. The aim of research was to investigate methanolic extracts from different parts (flowers, leaves, and stems) of Tunisian Rhanterium suaveolens as potential sources of bioactive products useful for healthy purposes. In particular, were analyzed the phenolic composition of these extracts and their antioxidant, anti-inflammatory, and anti-tyrosinase properties. The phytochemical analyses were performed using standard colorimetric procedures, HPLC-DAD and HPLC-DAD-ESI-MS. Then, several inâ vitro cell-free assays have been used to estimate the antioxidant/free radical scavenging capability of the extracts. Moreover, inâ vitro, and inâ vivo anti-melanogenesis activities of these extracts were tested, respectively, with the tyrosinase inhibition assay and the Zebrafish embryo model. Finally, the anti-inflammatory potential of these extracts in an inâ vitro model of acute intestinal inflammation in differentiated Caco-2 cells was evaluated. The R. suaveolens extracts under study appeared particularly rich in flavonols and hydroxycinnamic acids and all extracts appeared endowed with good antioxidant/free radical scavenging properties, being the flower extracts slightly more active than the others. Moreover, R. suaveolens flowers extract was able to inhibit inâ vitro tyrosinase activity and exhibited bleaching effects on the pigmentation of zebrafish embryos. Furthermore, all extracts showed good anti-inflammatory activity in intestinal epithelial cells as demonstrated by the inhibition of TNF-α-induced gene expression of IL-6 and IL-8. R. suaveolens aerial parts may be considered as a potential source of whitening agents, as well as of agents for the treatment of disorders related to oxidative stress and inflammation.