Isolation of flavonoids from Tagetes patula (French Marigold) flowers: Cytotoxic and oxidant activity in human cervical carcinoma cells / Aislación de flavonoides de las flores de Tagetes patula (cempasúchil francés): Actividad citotóxica y oxidante en células de carcinoma cervical humano

T agetes patula , known as French Marigold, belongs to the family Asteraceae. Human papillomavirus infection is considered one of the causes of cervical cancer. This study assessed the cytotoxic activity and intracellular oxidative capacity of compounds isolated from extract of T. patula flowers as anti - cancer cervical agents. Fraction F6 of n - butanol extract was subjected to column chromatography and HPLC - ESI - MS. The isolated compo unds of T. patula were used to examine cytotoxic activity and the production of total reactive oxygen species in SiHa and HeLa cells; the cells were also characterized using scanning electron microscopy. Patulitrin was cytotoxic to SiHa and HeLa cells. An increase in ROS production was observed at different times of treatment of cells with patuletin and patulitrin. Scanning electron microscopy showed morphological changes in SiHa and HeLa cells. Thus, compounds isolated from T. patula have great treatment p otential against cervical cancer.

Tagetes patula , conocida como cempasúchil francés, pertenece a la familia Asteraceae. La infección por el virus del papiloma humano se considera una de las causas del cáncer cervical. En este estudio, se evaluó la actividad citotóxica y la capacidad oxidativa intracelular de los compuestos aislados del extracto de las flores de T. patula como agentes anticancerígenos cervicales. La fracción F6 del ext racto de n - butanol se sometió a cromatografía en columna y HPLC - ESI - MS. Los compuestos aislados de T. patula se utilizaron para examinar la actividad citotóxica y la producción total de especies reactivas de oxígeno en las células SiHa y HeLa; las células también se caracterizaron mediante microscopía electrónica de barrido. Patulitrina resultó citotóxica para las células SiHa y HeLa. Se observó un aumento en la producción de ROS en diferentes momentos del tratamiento de las células con patuletina y patulit rina. La microscopía electrónica de barrido mostró cambios morfológicos en las células SiHa y HeLa. Por lo tanto, los compuestos aislados de T. patula tienen un gran potencial de tratamiento contra el cáncer cervical.

Dithiothreitol reduces oxidative stress and necrosis caused by ultraviolet A radiation in L929 fibroblasts.

Ultraviolet A (UVA) radiation, present in sunlight, can induce cell redox imbalance leading to cellular damage and even cell death, compromising skin health. Here, we evaluated the in vitro antioxidant and photochemoprotective effect of dithiothreitol (DTT). DTT neutralized the free radicals 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+), 2,2-diphenyl-1-picrylhydrazyl (DPPH·), and superoxide anion (O2·-) in in vitro assays, as well as the ferric ion (Fe3+) in the ferric reducing antioxidant power (FRAP) assay. We also evaluated the effect of DTT pre-treatment in L929 dermal fibroblasts and DTT (50 and 100 µM) led to greater cell viability following UVA-irradiation compared to cells that were untreated. Furthermore, the pre-treatment of cells with DTT prevented the increase of intracellular reactive oxygen species (ROS) production, including hydrogen peroxide (H2O2), lipid peroxidation, and DNA condensation, as well as the decrease in mitochondrial membrane potential (Δψm), that occurred following irradiation in untreated cells. The endogenous antioxidant system of cells was also improved in irradiated cells that were DTT pre-treated compared to the untreated cells, as the activity of the superoxide dismutase (SOD) and catalase (CAT) enzymes remained as high as non-irradiated cells, while the activity levels were depleted in the untreated irradiated cells. Furthermore, DTT reduced necrosis in UVA-irradiated fibroblasts. Together, these results showed that DTT may have promising use in the prevention of skin photoaging and photodamage induced by UVA, as it provided photochemoprotection against the harmful effects of this radiation, reducing oxidative stress and cell death, due mainly to its antioxidant capacity.

Berberine-fluconazole microparticle-based combination therapy to treat candidiasis infections.

AIM: This study aims to incorporate alginate microparticles containing berberine and fluconazole into two different types of pharmaceutical formulations, to subsequently evaluate the antifungal activity against Candida albicans. METHODS AND RESULTS: Alginate microparticles containing BBR (berberine) and FLU (fluconazole) were produced by the spray-drying technique, characterized and incorporated in two pharmaceutical formulations, a vaginal cream and artificial saliva. Broth microdilution, checkerboard, time-kill curve, and scanning electron microscopy were carried out to determine the antifungal effects of BBR and FLU against C. albicans. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of free BBR were 125 µg ml-1. Synergism between BBR and FLU was demonstrated by a fractional inhibitory concentration index (FICI) = 0.0762. The time-kill curve for the combination BBR + FLU showed a more pronounced decrease in fungal growth in comparison to free drugs, and an antibiofilm effect of BBR occurred in the formation and preformed biofilm. CONCLUSION: Alginate microparticles containing BBR and FLU were obtained and incorporated in a vaginal cream and artificial saliva. Both formulations showed good stability, antifungal effects, and organoleptic characteristics, which suggest that BBR-FLU microparticles in formulations have potential as antifungal therapy.

Advanced theranostic nanoplatforms for hypericin delivery in the cancer treatment.

Biomodified coated-lipid vesicles were obtained using the DPPC lipid (L) and F127 copolymer linked covalently with spermine (SN), biotin (BT), and folic acid (FA), resulting in LF127-SN, LF127-BT, and LF127-FA nanoplatforms. The photosensitizer hypericin (HY) was incorporated into the nanosystem by a thin-film method and characterized by dynamic light scattering, zeta potential, encapsulation efficiency, and transmission electronic microscopy. The results provided a good level of stability for all nanoplatforms for at least 5 days as an aqueous dispersion. The in vitro serum stability showed that the HY-loaded LF127-SN has a lower tendency to form complexes with BSA protein than with its analogs. LF127-SN was the most stable HY formulation, followed by LF127-BT and LF127-FA, confirmed by the association constant (Kd) values: 600 µmol L-1, 1100 µmol L-1, 515 µmol L-1, and 378 µmol L-1 for LF127, LF127 FA, LF127-BT, and LF127-SN, respectively. The photodynamic potential of HY was accessed by cytotoxicity assays using Caco-2, B16-F10, L-929, and HaCat cells. HY-loaded LF127-SN revealed a significant increase in the selectivity compared to other nanoplatforms. HY-loaded in LF127-BT and LF127-SN showed distinct uptake and biodistribution after 2 h of intravenous application. All biomodified coated-lipids showed satisfactory metabolism within 72 h after application, without significant accumulation or residue in any vital organ. These results suggest that incorporating HY-loaded in these nanosystems may be a promising strategy for future applications, even with a small amount of binders to the coating copolymer (0.02% w/v). Furthermore, these results indicate that the LF127-SN showed remarkable superiority compared to other evaluated systems, being the most distinct for future photodynamic therapy and theranostic applications.

Ionized alkaline water reduces injury in BALB/c mice infected with Leishmania amazonensis.

Ionized water has been reported to contribute to the tissue repair process and wound healing. Water purifiers can generate ionized water by means of activated charcoal with silver and minerals, the main purpose of which are to reduce microbiological and physicochemical contaminants. Moreover, when water is subjected to a magnetic field an organization of water molecules occurs due to the presence of mineral salts. The resulting water is thus more alkaline, which has been shown to be non-toxic to mice and can actually prolong survival. Cutaneous leishmaniasis is a neglected tropical disease, caused by obligate uni- and intracellular protozoa belonging to the genus Leishmania, that can manifest in the form of skin lesions. Thus, the objective of this study was to compare the evolution of disease in L. amazonensis-infected BALB/c mice that received tap water (TW) or ionized alkaline water (IAW). As a control, additional groups of mice receiving TW or IAW were also treated with the antileishmanial miltefosine. All mouse groups received either TW or IAW as drinking water 30 days prior to infection and the groups continued to receive the respective drinking water for 4 weeks, after which the blood and plasma were collected. Biochemical assays of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, creatinine, urea, glucose, triglycerides, and cholesterol were performed, in addition to hematology tests. There was a significant decrease in the volume of the lesion for groups that received IAW, in which the ingestion of ionized alkaline water favored the non-evolution of the lesion in the footpads of the animals. The results of the blood count and leukogram tests were within the normal values for BALB/c mice demonstrating that ionized water has no toxic effects on blood factors.

Antiproliferative Activity and Ultrastructural Changes in Promastigote and Amastigote forms of Leishmania amazonensis Caused by Limonene-Acylthiosemicarbazide Hybrids.

Leishmaniasis is a tropical zoonotic disease. It is found in 98 countries, with an estimated 1.3 million people being affected annually. During the life cycle, the Leishmania parasite alternates between promastigote and amastigote forms. The first line treatment for leishmaniasis are the pentavalent antimonials, such as N-methylglucamine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®). These drugs are commonly related to be associated with dangerous side effects such as cardiotoxicity, nephrotoxicity, hepatotoxicity, and pancreatitis. Considering these aspects, this work aimed to obtain a new series of limonene-acylthiosemicarbazides hybrids as an alternative for the treatment of leishmaniasis. For this, promastigotes, axenic amastigotes, and intracellular amastigotes of Leishmania amazonensis were used in the antiproliferative assay; J774-A1 macrophages for the cytotoxicity assay; and electron microscopy techniques were performed to analyze the morphology and ultrastructure of parasites. ATZ-S-04 compound showed the best result in both tests. Its IC50 , in promastigotes, axenic amastigotes and intracellular amastigotes was 0.35±0.08 µM, 0.49±0.06 µM, and 15.90±2.88 µM, respectively. Cytotoxicity assay determined a CC50 of 16.10±1.76 µM for the same compound. By electron microscopy, it was observed that ATZ-S-04 affected mainly the Golgi complex, in addition to morphological changes in promastigote forms of L. amazonensis.

ß-Carboline-α-aminophosphonate Derivative: A Promising Antitumor Agent for Breast Cancer Treatment.

Breast cancer is the most common type of cancer and the leading cause of cancer mortality among women worldwide. Considering the limitations of the current treatments available, we analyzed the in vitro cytotoxic potential of ((4-Fluoro-phenyl)-{2-[(1-phenyl-9H-ß-carboline-3-carbonyl)-amino]-ethylamino}-methyl)-phosphonic acid dibutyl ester (BCP-1) in breast cancer cells (MCF-7 and MDA-MB-231) and in a non-tumor breast cell line (MCF-10A). BCP-1 has an α-aminophosphonate unit linked to the ß-carboline nucleus, and the literature indicates that compounds of these classes have high biological potential. In the present study, the mechanism of action of BCP-1 was investigated through methods of spectrofluorimetry, flow cytometry, and protein expression analysis. It was found that BCP-1 inhibited the proliferation of both cancer cell lines. Furthermore, it induced oxidative stress and cell cycle arrest in G2/M. Upregulation of apoptosis-related proteins such as Bax, cytochrome C, and caspases, as well as a decrease in the anti-apoptotic protein Bcl-2, indicated potential induction of apoptosis in the MDA-MB-231 cells. While in MCF-7 cells, BCP-1 activated the autophagic death pathway, which was demonstrated by an increase in autophagic vacuoles and acidic organelles, in addition to increased expression of LC3I/LC3II and reduced SQSTM1/p62 expression. Further, BCP-1 demonstrated antimetastatic potential by reducing MMP-9 expression and cell migration in both breast cancer cell lines. In conclusion, BCP-1 is a promising candidate for breast cancer chemotherapy.

Synthetic dibenzylideneketones as promising anti-herpes simplex virus type 1 agents.

New antiviral agents for the treatment of herpes simplex virus type 1 (HSV-1) infection, which causes a highly prevalent and incurable disease, are needed. Here, we report for the first time the in vitro anti-HSV-1 activity of two dibenzylideneketone compounds: DBK1 and DBK2. DBK1 demonstrated virucidal activity, and high-resolution scanning electron microscopy showed that it caused morphological changes in the HSV-1 envelope. DBK2 was able to reduce HSV-1 plaque size in vitro. The DBKs are promising anti-HSV-1 candidates, as they exhibit low toxicity and exert an antiviral effect by acting at the early stages of HSV-1-host cell interaction.

Dibenzylideneacetone Induces Apoptosis in Cervical Cancer Cells through Ros-Mediated Mitochondrial Damage.

Cervical cancer is a health problem among women worldwide. Considering the limitations of prevention and antineoplastic chemotherapy against cervical cancer, research is needed to discover new, more effective, and safe antitumor agents. In the present study, we investigated the in vitro cytotoxicity of a new synthetic dibenzylideneacetone derived from 1,5-diaryl-3-oxo-1,4-pentadienyl (A3K2A3) against cervical cancer cells immortalized by HPV 16 (SiHa), and 18 (HeLa) by MTT assay. Furthermore, we performed spectrofluorimetry, flow cytometry, and Western blot analyzes to explore the inhibitory mechanism of A3K2A3 in cervical cancer cells. A3K2A3 showed cytotoxic activity against both cell lines. Mitochondrial depolarization and reduction in intracellular ATP levels were observed, which may be dependent on the redox imbalance between increased ROS and reduced levels of the antioxidant defense. In addition, damage to the cell membrane and DNA, and effective blocking of cell division in the G2/M phase were detected, which possibly led to the induction of apoptosis. This result was further confirmed by the upregulation of apoptosis-related proteins Bax, cytochrome C, and caspases 9 and 3. Our results provided the first evidence that A3K2A3 contributes to the suppression of cervical cancer in vitro, showing promise as a possible alternative for the treatment of this cancer.

Natural compounds based chemotherapeutic against Chagas disease and leishmaniasis: mitochondrion as a strategic target.

Over the past years, natural products have been explored in order to find biological active substances to treat various diseases. Regarding their potential action against parasites such as trypanosomatids, specially Trypanosoma cruzi and Leishmania spp., much advance has been achieved. Extracts and purified molecules of several species from genera Piper, Tanacetum, Porophyllum, and Copaifera have been widely investigated by our research group and exhibited interesting antitrypanosomal and antileishmanial activities. These natural compounds affected different structures in parasites, and we believe that the mitochondrion is a strategic target to induce parasite death. Considering that these trypanosomatids have a unique mitochondrion, this cellular target has been extensively studied aiming to find more selective drugs, since the current treatment of these neglected tropical diseases has some challenges such as high toxicity and prolonged treatment time. Here, we summarise some results obtained with natural products from our research group and we further highlighted some strategies that must be considered to finally develop an effective chemotherapeutic agent against these parasites.

ß-carbolines RCC and C5 induce the death of Leishmania amazonensis intracellular amastigotes.

Background: Cutaneous leishmaniasis is caused by Leishmania spp., and its treatment is limited. The ß-carbolines have shown activity against kinetoplastids. Aim: To evaluate the activity and effects of the ß-carbolines, N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-methoxyphenyl)-ß-carboline-3-carboxamide (RCC) and N-benzyl-1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide (C5), against L. amazonensis intracellular amastigotes and to suggest their mechanism of action. Methods: We analyzed the activity and cytotoxicity of ß-carbolines and the morphological alterations by electron microscopy. Mitochondrial membrane potential, production nitric oxide, reactive oxygen species, lipidic bodies, autophagic vacuoles and ATP were also evaluated. Results & conclusion: The results showed that RCC and C5 are active against intracellular amastigotes and were able to induce oxidative stress and ultrastructural alterations such as accumulation of lipid bodies and autophagic vacuoles, leading to parasite death.

Multifunctional Nanoparticles as High-Efficient Targeted Hypericin System for Theranostic Melanoma.

Biotin, spermine, and folic acid were covalently linked to the F127 copolymer to obtain a new drug delivery system designed for HY-loaded PDT treatment against B16F10 cells. Chemical structures and binders quantification were performed by spectroscopy and spectrophotometric techniques (1NMR, HABA/Avidin reagent, fluorescamine assay). Critical micelle concentration, critical micelle temperature, size, polydispersity, and zeta potential indicate the hydrophobicity of the binders can influence the physicochemical parameters. Spermine-modified micelles showed fewer changes in their physical and chemical parameters than the F127 micelles without modification. Furthermore, zeta potential measurements suggest an increase in the physical stability of these carrier systems. The phototherapeutic potential was demonstrated using hypericin-loaded formulation against B16F10 cells, which shows that the combination of the binders on F127 copolymer micelles enhances the photosensitizer uptake and potentializes the photodynamic activity.

Development and evaluation of topical formulations that contain hydroethanolic extract from Schinus terebinthifolia against HSV-1 infection

Abstract The therapeutic drugs to treat Herpes simplex virus (HSV) infections have toxic side effects and there has been an emergence of drug-resistant strains. Therefore, the search for new treatments for HSV infections is mounting. In the present study, semi-solid formulations containing a crude hydroethanolic extract (CHE) from Schinus terebinthifolia were developed. Skin irritation, cutaneous permeation, and in vivo therapeutic efficacy of the formulations were investigated. Treatment with the ointment formulations did not result in any signs of skin irritation while the emulsions increased the thickness of the epidermis in Swiss mice. The cutaneous permeation test indicated that the CHE incorporated in the formulations permeated through the skin layers and was present in the epidermis and dermis even 3 h after topical application. In vivo antiviral activity in BALB/c mice treated with the CHE ointments was better than those treated with the CHE emulsions and did not significantly differ from an acyclovir-treated group. Taken together, this suggests that the incorporation of CHE in the ointment may be a potential candidate for the alternative topical treatment of herpetic lesions.

Natural compounds based chemotherapeutic against Chagas disease and leishmaniasis: mitochondrion as a strategic target

Over the past years, natural products have been explored in order to find biological active substances to treat various diseases. Regarding their potential action against parasites such as trypanosomatids, specially Trypanosoma cruzi and Leishmania spp., much advance has been achieved. Extracts and purified molecules of several species from genera Piper, Tanacetum, Porophyllum, and Copaifera have been widely investigated by our research group and exhibited interesting antitrypanosomal and antileishmanial activities. These natural compounds affected different structures in parasites, and we believe that the mitochondrion is a strategic target to induce parasite death. Considering that these trypanosomatids have a unique mitochondrion, this cellular target has been extensively studied aiming to find more selective drugs, since the current treatment of these neglected tropical diseases has some challenges such as high toxicity and prolonged treatment time. Here, we summarise some results obtained with natural products from our research group and we further highlighted some strategies that must be considered to finally develop an effective chemotherapeutic agent against these parasites.

Herpes Labialis: A New Possibility for Topical Treatment with Well-Elucidated Drugs.

Mucocutaneous infections caused by Herpes simplex virus (HSV-1 and HSV-2) are characterized by the appearance of vesicles that cause pain and embarrassment to the carrier. The standard treatment is based on the use of antivirals in gels or ointments, however, relapses are common. Local anesthetics decrease the pain caused by the lesion, in addition to showing antiviral properties. Semi-solid form facilitates application and its transformation into a thin film favors the maintenance of the formulation in place, with a more discreet final aspect. The objective of this study was to develop and evaluate formulations containing anesthetics for the treatment of cold sores. For this purpose, two semi-solid film-forming formulations were developed and evaluated, containing HPMC K100, lidocaine (LIDO) and prilocaine (PRILO) combined with adjuvants, in the presence (F1T) or not (F1) of the absorption promoter Transcutol®. The mixture of PRILO and LIDO resulted in the formation of a eutectic mixture (EM), essential for penetration of drugs into the skin. The quantification of drugs was performed by HPLC (High Performance Liquid Chromatography), and Transcutol® did not influence the release of drugs from the formulation. The bioadhesiveness of the formulation was evaluate and the drugs did not impair the adhesive potential of the polymers used. The formulations were evaluated in vivo for skin irritation and did not show any negative sign on macroscopic examination. The in vivo efficacy test proved the anesthetics' ability to decrease the lesions caused by HSV-1. Thus, the proposed formulations proved to be good alternatives to the treatment of oral lesions caused by HSV-1.

Membrane dynamics in Leishmania amazonensis and antileishmanial activities of ß-carboline derivatives.

Two ß-carboline compounds, 8i and 6d, demonstrated in vitro antileishmanial activity against Leishmania (L.) amazonensis promastigotes similar to that of miltefosine (MIL). Estimates of the membrane-water partition coefficient (KM/W) and the compound concentrations in the membrane (cm50) and aqueous phase (cw50) for half maximal inhibitory concentration were made. Whereas these biophysical parameters for 6d were not significantly different from those reported for MIL, 8i showed lower affinity for the parasite membrane (lower KM/W) and a lower concentration of the compound in the membrane required to inhibit the growth of the parasite (lower cm50). A 2-hour treatment of Leishmania promastigotes with the compounds 8i and 6d caused membrane rigidity in a concentration-dependent manner, as demonstrated by the electron paramagnetic resonance (EPR) technique and spin label method. This increased rigidity of the membrane was interpreted to be associated with the occurrence of cross-linking of oxidized cytoplasmic proteins to the parasite membrane skeleton. Importantly, the two ß-carboline-oxazoline derivatives showed low hemolytic action, both in experiments with isolated red blood cells or with whole blood, denoting their great Leishmania/erythrocyte selectivity index. Using electron microscopy, changes in the membrane of both the amastigote and promastigote form of the parasite were confirmed, and it was demonstrated that compounds 8i and 6d decreased the number of amastigotes in infected murine macrophages. Furthermore, 8i and 6d were more toxic to the protozoa than to J774A.1 macrophages, with treated promastigotes exhibiting a decrease in cell volume, mitochondrial membrane potential depolarization, accumulation of lipid bodies, increased ROS production and changes in the cell cycle.

Ceria Nanoparticles Decrease UVA-Induced Fibroblast Death Through Cell Redox Regulation Leading to Cell Survival, Migration and Proliferation.

Exposure to ultraviolet radiation is a major contributor to premature skin aging and carcinogenesis, which is mainly driven by overproduction of reactive oxygen species (ROS). There is growing interest for research on new strategies that address photoaging prevention, such as the use of nanomaterials. Cerium oxide nanoparticles (nanoceria) show enzyme-like activity in scavenging ROS. Herein, our goal was to study whether under ultraviolet A rays (UVA)-induced oxidative redox imbalance, a low dose of nanoceria induces protective effects on cell survival, migration, and proliferation. Fibroblasts cells (L929) were pretreated with nanoceria (100 nM) and exposed to UVA radiation. Pretreatment of cells with nanoceria showed negligible cytotoxicity and protected cells from UVA-induced death. Nanoceria also inhibited ROS production immediately after irradiation and for up to 48 h and restored the superoxide dismutase (SOD) activity and GSH level. Additionally, the nanoceria pretreatment prevented apoptosis by decreasing Caspase 3/7 levels and the loss of mitochondrial membrane potential. Nanoceria significantly improved the cell survival migration and increased proliferation, over a 5 days period, as compared with UVA-irradiated cells, in wound healing assay. Furthermore, it was observed that nanoceria decreased cellular aging and ERK 1/2 phosphorylation. Our study suggests that nanoceria might be a potential ally to endogenous, antioxidant enzymes, and enhancing the redox potentials to fight against UVA-induced photodamage and consequently modulating the cells survival, migration, and proliferation.

Metformin effect on driving cell survival pathway through inhibition of UVB-induced ROS formation in human keratinocytes.

Human skin functions go beyond serving only as a mechanical barrier. As a complex organ, the skin is capable to cope with external stressors cutaneous by neuroendocrine systems to control homeostasis. However, constant skin exposure to ultraviolet (UV) radiation causes progressive damage to cellular skin constituents, mainly due excessive reactive oxygen species (ROS) production. The present study shows new approaches of metformin (MET) as an antioxidant agent. Currently, MET is the first line treatment of type 2 diabetes and has attracted attention, based on its broad mechanism of action. Therefore, we evaluated MET antioxidant potential in cell-free systems and in UVB irradiated human keratinocyte HaCaT cells. In cell-free system assays MET did not show intrinsic scavenging activity on DPPH radicals or superoxide (O2-) xanthine/luminol/xanthine oxidase-generated. Cell-based results demonstrated that MET was able to reduce UVB-induced intracellular ROS and NADPH oxidase-dependent superoxide (O2-) production. MET posttreatment of HaCaT cells reduced ERK 1/2 phosphorylation, NADPH oxidase activity, and cell death by apoptosis. These findings suggest that the protection mechanism of MET may be through the inhibition of ROS formation enzyme. These results showed that MET might be a promising antioxidant agent against UV radiation induced skin damage.

Antiproliferative activity of the dibenzylideneacetone derivate (E)-3-ethyl-4-(4-nitrophenyl)but­3-en-2-one in Trypanosoma cruzi.

Chagas disease is one of the most prevalent neglected diseases in the world. The illness is caused by Trypanosoma cruzi, a protozoan parasite with a complex life cycle and three morphologically distinct developmental stages. Nowadays, the only treatment is based on two nitro-derivative drugs, benznidazole and nifurtimox, which cause serious side effects. Since the treatment is limited, the search for new treatment options for patients with Chagas disease is highly necessary. In this study we analyzed the substance A11K3, a dibenzylideneacetone (DBA). DBAs have an acyclic dienone attached to aryl groups in both ß-positions and studies have shown that they have biological activity against tumors cells, bacteria, and protozoa such as T. cruzi and Leishmania spp. Here we show that A11K3 is active against all three T. cruzi evolutionary forms: the epimastigote (IC50 = 3.3 ± 0.8), the trypomastigote (EC50 = 24 ± 4.3) and the intracellular amastigote (IC50 = 9.3 ± 0.5 µM). A cytotoxicity assay in LLCMK2 cells showed a CC50 of 239.2 ± 15.7 µM giving a selectivity index (CC50/IC50) of 72.7 for epimastigotes, 9.9 for trypomastigotes and 25.9 for intracellular amastigotes. Morphological and ultrastructural analysis of the parasites treated with A11K3 by TEM and SEM revealed alterations in the Golgi complex, mitochondria, plasma membrane and cell body, with an increase of autophagic vacuoles and lipid bodies. Biochemical assays of A11K3-treated T. cruzi showed an increase of ROS, plasma membrane ruptures, lipid peroxidation, mitochondrial membrane depolarization with a decrease in ATP and accumulation of autophagic vacuoles. The results lead to the hypothesis that A11K3 causes death of the protozoan through events such as plasma membrane and mitochondrial alterations and autophagy, characteristic of cell collapse.

Development of chitosan nanocapsules containing essential oil of Matricaria chamomilla L. for the treatment of cutaneous leishmaniasis.

Matricaria chamomilla L. has been used for centuries in many applications, including antiparasitic activity. Leishmaniasis is a parasitic disease, with limited treatments, due to high cost and toxicity. Thus, there is a need to develop new treatments, and in this context, natural products are targets of these researches. We report the development of chitosan nanocapsules containing essential oil of M. chamomilla (CEO) from oil-in-water emulsions using chitosan modified with tetradecyl chains as biocompatible shell material. The nanocapsules of CEO (NCEO) were analyzed by optical microscopy and dynamic light scattering, which revealed spherical shape and an average size of 800 nm. Successful encapsulation of CEO was further confirmed by fluorescence microscopy observations taking advantage of the autofluorescence properties of CEO. The encapsulation efficiency was around 90%. The entrapment of CEO reduced its cytotoxicity towards normal cells. On the other hand, the CEO was active against promastigotes and intracellular amastigotes, exhibiting IC50 of 3.33 µg/mL and 14.56 µg/mL, respectively, while NCEO showed IC50 for promastigotes of 7.18 µg/mL and for intracellular amastigotes of 14.29 µg/mL. These results demonstrate that encapsulation of CEO in nanocapsules using an alkylated chitosan biosurfactant as a "green" stabilizer is a promising therapeutic strategy to treat leishmaniasis.