Copaiba Oil Resin Exerts an Additive Effect to Babassu Oil on Behavioral Changes in Human Endometriotic Cell Cultures.

BACKGROUND: Current drugs for the treatment of endometriosis are not able to completely cure the condition, and significant side effects hinder the continuation of treatment. Therefore, it is necessary to search for new drug candidates. In the present paper, the use of plant extracts is highlighted. Babassu oil and Copaiba oil resin have several therapeutic properties. We investigated the in vitro effects of two nanoemulsions containing oil extracted from Babassu (Orbignya speciosa) nuts (called SNEDDS-18) and/or oil resin extracted from Copaiba trunk (Copaifera langsdorffii) (called SNEDDS-18/COPA) on cultured human eutopic endometrium stromal cells from endometrial biopsies of patients without (CESC) and with (EuESC) endometriosis as well as human stromal cells from biopsies of endometriotic lesions (EctESC). METHODS: CESC, EuESC, and EctESC were taken and treated with SNEDDS-18 and SNEDDS-18/COPA to evaluate their effects on cytotoxicity, cell morphology, proliferation, and signaling pathways. RESULTS: After 48 h of incubation with SNEDDS-18 and SNEDDS-18/COPA, cell viability and proliferation were inhibited, especially in EctESC. The lowest concentration of both nanoemulsions reduced cell viability and proliferation and broke down the cytoskeleton in EctESCs. After 24 h of treatment a decrease in IL-1, TNF-α, and MCP-1 was observed, as well as an increase in IL-10 production. CONCLUSIONS: Both nanoemulsions can affect endometriotic stromal cell behaviors, thus revealing two potential candidates for new phytotherapeutic agents for the management of endometriosis.

Antimycobacterial and anti-inflammatory activities of thiourea derivatives focusing on treatment approaches for severe pulmonary tuberculosis.

Tuberculosis (TB) remains a serious public health problem and one of the main concern is the emergence of multidrug-resistant and extensively resistant TB. Hyper-reactive patients develop inflammatory necrotic lung lesions that aggravate the pathology and facilitate transmission of mycobacteria. Treatment of severe TB is a major clinical challenge that has few effective solutions and patients face a poor prognosis, years of treatment and different adverse drug reactions. In this work, fifteen novel and thirty-one unusual thiourea derivatives were synthesized and evaluated in vitro for their antimycobacterial and anti-inflammatory potential and, in silico for ADMET parameters and for structure-activity relationship (SAR). Thioureas derivatives 10, 15, 16, 28 and 29 that had shown low cytotoxicity and high activities were selected for further investigation, after SAR study. These five thioureas derivatives inhibited Mtb H37Rv growth in bacterial culture and in infected macrophages, highlighting thiourea derivative 28 (MIC50 2.0 ± 1.1 and 2.3 ± 1.1 µM, respectively). Moreover, these compounds were active against the hypervirulent clinical Mtb strain M299, in bacterial culture, especially 16, 28 and 29, and in extracellular clumps, highlighting 29, with MIC50 5.6 ± 1.2 µM. Regarding inflammation, they inhibited NO through the suppression of iNOS expression, and also inhibited the production of TNF-α and IL-1ß. In silico studies were carried out suggesting that these five compounds could be administered by oral route and have low toxicological effects when compared to rifampicin. In conclusion, our data show that, at least, thiourea derivatives 16, 28 and 29 are promising antimycobacterial and anti-inflammatory agents, and candidates for further prospective studies aiming new anti-TB drugs, that can be used on a dual approach for the treatment of severe TB cases associated with exacerbated inflammation.

Development of rivaroxaban microemulsion-based hydrogel for transdermal treatment and prevention of venous thromboembolism.

We have developed a microemulsion (ME)-based hydrogel, containing propylene glycol, Azone®, Labrasol®, isobutanol and water (20:3:18:3:56), for the transdermal delivery of rivaroxaban (RVX). Formulation ME-1:RVX, which was loaded with 0.3 mg/g of RVX, presented as a clear, homogenous fluid with a droplet size of 82.01 ± 6.32 nm and a PdI of 0.207 ± 0.01. To provide gelation properties, 20 % (w/w) of Pluronic® F-127 was added to ME-1:RVX to generate formulation PME-1a. An added benefit was an increased capacity for RVX to 0.4 mg/g (formulation PME-1b). PME-1b displayed spherical droplets with a nanoscale diameter as observed by Transmission Electron Microscopy. The release of RVX from PME-1b was 20.71 ± 0.76 µg/cm2 with a permeation through pig epidermis of 18.32 ± 8.87 µg/cm2 as measured in a Franz Cell for 24 h. PME-1b presented a pseudoplastic behavior, pH value compatible with the skin and good stability over 60 days at room and elevated temperatures. The prothrombin time was assessed for each concentration of RVX obtained in the permeation assay and each demonstrated a relevant anticoagulant activity. PME-1b also presented no cytotoxicity against HaCaT cells. Utilizing GastroPlus® software, an in silico analysis was performed to simulate the delivery of PME-1b through a transdermal system that suggested a minimum dose of RVX for the treatment and prevention of venous thromboembolism could be achieved with an 8 h administration regimen. These results suggest that PME-1b is a promising transdermal formulation for the effective delivery of RVX that could be a viable alternative for the treatment and prevention of venous thromboembolism.

Laser therapy increases the proliferation of preosteoblastic MC3T3-E1 cells cultured on poly(lactic acid) films.

This study aimed to verify the efficacy of low-level laser irradiation (LLLI) on the proliferation of MC3T3-E1 preosteoblasts cultured on poly(lactic acid) (PLA) films. The produced films were characterized by contact angle tests, scanning electron microscopy (SEM), atomic force microscopy, differential scanning calorimetry, and X-ray diffraction. The MC3T3-E1 cells were cultured as three different groups: Control-cultured on polystyrene plastic surfaces; PLA-cultured on PLA films; and PLA + Laser-cultured on PLA films and submitted to laser irradiation (660 nm; 30 mW; 4 J/cm2 ). Cell proliferation was analyzed by Trypan blue and Alamar blue assays at 24, 48, and 72 h after irradiation. Cell viability was assessed by Live/Dead assay, apoptosis-related events were evaluated by Annexin V/propidium iodide (PI) expression, and cell cycle events were analyzed by flow cytometry. Cell morphology on the surface of films was assessed by SEM. Cell counting and biochemical assay results indicate that the PLA + Laser group exhibited higher proliferation (p < 0.01) when compared with the Control and PLA groups. The Live/Dead and Annexin/PI assays indicate increased cell viability in the PLA + Laser group that also presented a higher percentage of cells in the proliferative cell cycle phases (S and G2/M). These findings were also confirmed by the higher cell density observed in the irradiated group through SEM images. The evidence from this study supports the idea that LLLI increases the proliferation of MC3T3-E1 cells on PLA surfaces, suggesting that it can be potentially applied in bone tissue engineering.

Development and characterization of poly(lactic-co-glycolic) acid nanoparticles loaded with copaiba oleoresin.

Copaiba oleoresin (CPO), obtained from Copaifera landgroffii, is described as active to a large number of diseases and more recently in the endometriosis treatment. In this work, poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing CPO were obtained using the design of experiments (DOE) as a tool to optimize the production process. The nanoparticles optimized by means of DOE presented an activity in relation to the cellular viability of endometrial cells. The DOE showed that higher amounts of CPO combined with higher surfactant concentrations resulted in better encapsulation efficiency and size distribution along with good stability after freeze drying. The encapsulation efficiency was over 80% for all produced nanoparticles, which also presented sizes below 300 nm and spherical shape. A decrease in viability of endometrial stromal cells from ectopic endometrium of patients with endometriosis and from eutopic endometriotic lesions was demonstrated after 48 h of incubation with the CPO nanoparticles. The nanoparticles without CPO were not able to alter the cell viability of the same cells, indicating that this material was not cytotoxic to the tested cells and suggesting that the effect was specific to CPO. The results indicate that the use of CPO nanoparticles may represent a promising alternative for the treatment of endometriosis.

Development and pharmacological evaluation of in vitro nanocarriers composed of lamellar silicates containing copaiba oil-resin for treatment of endometriosis.

In this work, newly developed nanocomposites based upon lamellar silicates are evaluated to determine their potential in controlling endometriosis. The preparation of the new nanocarriers is detailed, properties characterized and in vitro pharmacological evaluation performed. The nanocomposites in this study were obtained from the reaction of copaiba oil-resin (COPA) with the polymer polyvinylpyrrolidone (PVP K-30). COPA was selected due to its antiinflammatory and anticancer activities along with the organophilic derivatives of sodium montmorillonite, Viscogel B8, S7 and S4. The results indicated that it was feasible to obtain a good yield of a COPA nanocomposite using a simple process. Intercalation was confirmed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). In vitro release experiments demonstrated that COPA was released from the nanocomposite in a delayed fashion. Whereas, in vitro pharmacological studies showed a reduction in viability and proliferation of endometriotic cell cultures upon COPA nanocomposite treatment, suggesting that the system developed here can be a promising alternative therapy for the oral treatment of endometriosis.

The oil-resin of the tropical rainforest tree Copaifera langsdorffii reduces cell viability, changes cell morphology and induces cell death in human endometriotic stromal cultures.

OBJECTIVES: The hormonal treatment for endometriosis frequently fails to completely eradicate endometriotic implants. A new therapeutic treatment is needed. This study investigates the in-vitro effect of Copaifera langsdorffii oil-resin on human eutopic and ectopic endometrium stromal cell cultures (EuESCs and EctESCs). METHODS: A nanocomposite system containing the copaiba oil-resin (NanoCOR) was developed and acute toxicity test was performed. Endometrial stromal cells (ESCs) from non-endometriotics controls (CESCs), EuESCs and EctESCs were isolated and treated with different concentrations of NanoCOR, at different time intervals to evaluate its effect on cell morphology, proliferation, viability, necrosis and apoptosis induction. KEY FINDINGS: When treated with 50 µg/ml of NanoCOR, the morphology of EctESCs changed, as the actin microfilaments were disorganized, disassembled or disrupted. Moreover, at 24 h of treatment with NanoCOR, the EctESCs viability was inhibited, and a significant number of these cells underwent apoptosis. In EuESCs, these effects were observed only at 48 h. Finally, the treatment of EctESCs with NanoCOR increased the lactate dehydrogenase release into the extracellular medium more than in EuESCs. CONCLUSIONS: Our data indicate that NanoCOR has a greater impact on the behaviour of human endometriotic stromal cells than on the eutopic endometrium stromal cells, supporting the idea that NanoCOR should be further investigated as a novel and valuable alternative to treat endometriosis.

Acaricidal properties of the formulations based on essential oils from Cymbopogon winterianus and Syzygium aromaticum plants.

The cattle tick, Rhipicephalus (Boophilus) microplus, has caused serious harm to livestock raising in Brazil, considering the costs of controlling it, loss of revenue due to smaller production of milk and meat, and damage to leather, in addition to transmitting diseases. The use of medicinal plants is considered an alternative to the recurring resistance to chemicals. Due to the need for efficient alternatives with less environmental impact, this study aimed to develop contact formulations with essential oils from the Java citronella (Cymbopogon winterianus) and clove (Syzygium aromaticum) plants and to assess in vitro the effects in different stages of the tick cycle. In the present study, concentrations from 0.5-15.0% of the essential oils incorporated in the formulations were used. The ticks from different geographical areas were treated with those formulations, and their effects on the production levels of eggs, on the larvae hatching, and their efficiency on ticks were assessed. The obtained results were compared with other commercial acaricidal products. After the 20th day of treatment, the formulations with citronella essential oil had 2.09-55.51% efficiency, depending on the concentration of the oil incorporated. The efficiency of the treatment with formulations containing clove essential oil was higher, from 92.47-100%. The results showed the acaricidal effects of the formulations tested when compared to commercial chemical products. In vivo studies should be performed in order to assess the efficiency of those formulations in the fields, aiming to use these products as an alternative for controlling cattle ticks.

Development of a high performance liquid chromatography method for quantification of isomers ß-caryophyllene and α-humulene in copaiba oleoresin using the Box-Behnken design.

The sesquiterpene isomers, ß-Cariofileno (CAR) and α-Humuleno (HUM) are the primary constituents of the copaiba oleoresin species. These natural products are primarily used by the Amazonian population and marketed as phytotherapies and cosmetics. The aim of this study was to develop and validate a method that simultaneously assays the isomers present in copaiba oleoresins by high performance liquid chromatography using the Box-Behnken design. After preliminary studies, the reverse phase chromatographic system was selected using a cyano column and a mobile phase consisting of acetonitrile and phosphate buffer. The Box-Behnken design was applied at three levels and with four independent variables: flow rate (X1), gradient slope time (X2), proportion of organic compounds at the end of the gradient (X3) and at the beginning of the gradient (X4). Also, the responses of the dependent variables: CAR retention time (Y1) and the resolution between the CAR and HUM peaks (Y2) was assessed. The mathematical model obtained from the regression results was satisfactory (R(2)>0.98, n=27) and showed a quadratic relationship where the effects of interactions between the variables, was observed by response surface graphs. The simultaneous optimization method was used to establish the best compromise of the resolution between the CAR and HUM isomers while adjusting the retention time of CAR. This method was successfully optimized by BBD obtaining chromatographic peaks with good symmetry, resolution and separation efficiency. The validation of the developed method confirmed its specificity, precision, accuracy and linearity in the range of 5.0-11.0 and 0.4-1.0µg/mL for CAR and HUM, respectively, and is considered suitable for routine applications which assure quality control.

Nanostructured systems containing babassu (Orbignya speciosa) oil as a potential alternative therapy for benign prostatic hyperplasia.

The oil of babassu tree nuts (Orbignya speciosa) is a potential alternative for treatment and prophylaxis of benign prostatic hyperplasia. Improved results can be obtained by drug vectorization to the hyperplastic tissue. The main objective of this work was the preparation and characterization of poly(lactic-co-glycolic acid) (PLGA) nanoparticle and clay nanosystems containing babassu oil (BBS). BBS was extracted from the kernels of babassu tree nuts and characterized by gas chromatography-mass spectrometry as well as 1H and 13C nuclear magnetic resonance. BBS-clay nanosystems were obtained by adding polyvinylpyrrolidone, Viscogel B8®, and BBS at a 2:1:1 mass ratio and characterized by X-ray diffraction, thermogravimetric analysis, infrared spectroscopy, and laser diffraction. The PLGA-BBS nanoparticles were prepared by the precipitation-solvent evaporation method. Mean diameter, polydispersity, zeta potential, and scanning electron microscopic images of the nanosystems were analyzed. Thermogravimetric analysis showed successful formation of the nanocomposite. PLGA nanoparticles containing BBS were obtained, with a suitable size that was confirmed by scanning electron microscopy. Both nanostructured systems showed active incorporation yields exceeding 90%. The two systems obtained represent a new and potentially efficient therapy for benign prostatic hyperplasia.

Poly ɛ-caprolactone nanoparticles loaded with Uncaria tomentosa extract: preparation, characterization, and optimization using the Box-Behnken design.

PURPOSE: The aim of this research was to develop and optimize a process for obtaining poly ɛ-caprolactone (PCL) nanoparticles loaded with Uncaria tomentosa (UT) extract. METHODS: Nanoparticles were produced by the oil-in-water emulsion solvent evaporation method. Preliminary experiments determined the initial conditions of the organic phase (OP) and of the aqueous phase (AP) that would be utilized for this study. Ultimately, a three-factor three-level Box-Behnken design (BBD) was employed during the optimization process. PCL and polyvinyl alcohol (PVA) concentrations (X(1) and X(2), respectively) and the AP/OP volume ratio (X(3)) were the independent variables studied, while entrapment efficiency (Y(1)), particle mean diameter (Y(2)), polydispersity (Y(3)), and zeta potential (Y(4)) served as the evaluated responses. RESULTS: PRELIMINARY EXPERIMENTS REVEALED THAT THE OPTIMAL INITIAL CONDITIONS FOR THE PREPARATION OF NANOPARTICLES WERE AS FOLLOWS: OP composed of 5 mL ethyl acetate/acetone (3/2) mixture containing UT extract and PCL, and an AP of buffered PVA (pH 7.5) solution. Statistical analysis of the BBD results indicated that all of the studied factors had significant effects on the responses Y(1), Y(2), and Y(4,) and these effects are closely described or fitted by regression equations. Based on the obtained models and the selected desirability function, the nanoparticles were optimized to maximize Y(1) and minimize Y(2). These optimal conditions were achieved using 3% (w/v) PCL, 1% (w/v) PVA, and an AP/OP ratio of 1.7, with predicted values of 89.1% for Y(1) and 280 nm for Y(2). Another batch was produced under the same optimal conditions. The entrapment efficiency of this new batch was measured at 81.6% (Y(1)) and the particles had a mean size of 247 nm (Y(2)) and a polydispersity index of 0.062 (Y(3)). CONCLUSION: This investigation obtained UT-loaded nanoparticle formulations with desired characteristics. The BBD approach was a useful tool for nanoparticle development and optimization, and thus should be useful especially in the realm of phytotherapeutics, in which varied compositions may be assessed in quantitative and qualitative terms.

Influence of the calcium concentration in the presence of organic phosphorus on the physicochemical compatibility and stability of all-in-one admixtures for neonatal use.

BACKGROUND: Preterm infants need high amounts of calcium and phosphorus for bone mineralization, which is difficult to obtain with parenteral feeding due to the low solubility of these salts. The objective of this study was to evaluate the physicochemical compatibility of high concentrations of calcium associated with organic phosphate and its influence on the stability of AIO admixtures for neonatal use. METHODS: Three TPN admixture formulas were prepared in multilayered bags. The calcium content of the admixtures was adjusted to 0, 46.5 or 93 mg/100 ml in the presence of a fixed organic phosphate concentration as well as lipids, amino acids, inorganic salts, glucose, vitamins and oligoelements at pH 5.5. Each admixture was stored at 4 degrees C, 25 degrees C or 37 degrees C and evaluated over a period of 7 days. The physicochemical stability parameters evaluated were visual aspect, pH, sterility, osmolality, peroxide formation, precipitation, and the size of lipid globules. RESULTS: Color alterations occurred from the first day on, and reversible lipid film formation from the third day of study for the admixtures stored at 25 degrees C and 37 degrees C. According to the parameters evaluated, the admixtures were stable at 4 degrees C; and none of them presented precipitated particles due to calcium/phosphate incompatibility or lipid globules larger than 5 mum, which is the main parameter currently used to evaluate lipid emulsion stability. The admixtures maintained low peroxide levels and osmolarity was appropriate for parenteral administration. CONCLUSION: The total calcium and calcium/phosphorus ratios studied appeared not to influence the physicochemical compatibility and stability of AIO admixtures.

Avaliação da adequação técnica de indústrias de medicamentos fitoterápicos e oficinais do Estado do Rio de Janeiro / Assessment of the technical adequacy of phytotherapeutics and officinal medicine manufacturers in the State of Rio de Janeiro

Neste trabalho, buscou-se determinar o perfil de indústrias farmacêuticas dedicadas à fabricação e fracionamento de medicamentos fitoterápicos e oficinais no Estado do Rio de Janeiro por informações do CVS-RJ. Foram identificadas 48 empresas com atividades relacionadas à fabricação e/ou fracionamento e distribuição de medicamentos oficinais e fitoterápicos e analisadas no grau de adequação à legislação sanitária vigente, cumprimento de boas práticas de fabricação vigentes, bem como às questões relacionadas ao registro de produtos; as principais irregularidades foram o fluxo de pessoal e controle de qualidade adequado. Constatou-se que há atualmente empresas em situação: satisfatória (29,2 por cento), satisfatórias com restrições (10,4 por cento), insatisfatórias (6,2 por cento), interditadas (39,6 por cento), e solicitantes do cancelamento do processo por não terem condições para o cumprimento da RDC nº 210/03 (14,6 por cento). Segundo o quadro atual, ainda é grande o número de empresas com atividades relacionadas à fabricação e/ou fracionamento e distribuição de medicamentos fitoterápicos e oficinais em fase de adequação ou em condições não adequadas no Rio de Janeiro. Ao mesmo tempo, é pouco factível a adequação, frente ao tipo de produto e porte financeiro, apontando um prognóstico desfavorável para o setor em questão.

In this study we evaluated the current profile of manufacturers of officinal medicines and phytotherapeutics in relation to the Brazilian regulatory legislation. Forty-eight industries involved in manufacture and distribution of officinal medicines and phytotherapeutics were identified in one hundred current administrative processes of the National Health Surveillance Agency in Brazil. The analysis of the inspection reports of these companies considered the first nine months after the implantation of Resolution RDC 210/03 and revealed five specific company profiles: satisfactory (29.2 percent), satisfactory with restrictions (10.4 percent), unsatisfactory (6.2 percent), interdicted (39.6 percent), and applying for cancellation due to lack of conditions for complying with Resolution RDC 210/03 (14.6 percent). The main irregularities found in these companies involved the operational flow, quality control and product registration issues. Our results revealed a great number of companies whose activities are related to the production of officinal medicines and phytotherapeutics but that are still not totally adequate to the industrial park of Rio de Janeiro. The perspectives for the future of this category of industries are thus not favorable because of their difficulties to meet the current requirements.

[Assessment of the technical adequacy of phytotherapeutics and officinal medicine manufacturers in the State of Rio de Janeiro]. / Avaliação da adequação técnica de indústrias de medicamentos fitoterápicos e oficinais do Estado do Rio de Janeiro.

In this study we evaluated the current profile of manufacturers of officinal medicines and phytotherapeutics in relation to the Brazilian regulatory legislation. Forty-eight industries involved in manufacture and distribution of officinal medicines and phytotherapeutics were identified in one hundred current administrative processes of the National Health Surveillance Agency in Brazil. The analysis of the inspection reports of these companies considered the first nine months after the implantation of Resolution RDC 210/03 and revealed five specific company profiles: satisfactory (29.2%), satisfactory with restrictions (10.4%), unsatisfactory (6.2%), interdicted (39.6%), and applying for cancellation due to lack of conditions for complying with Resolution RDC 210/03 (14.6%). The main irregularities found in these companies involved the operational flow, quality control and product registration issues. Our results revealed a great number of companies whose activities are related to the production of officinal medicines and phytotherapeutics but that are still not totally adequate to the industrial park of Rio de Janeiro. The perspectives for the future of this category of industries are thus not favorable because of their difficulties to meet the current requirements.