Anticoagulant and antiplatelet activity of aqueous extracts of Citrus sinensis and Lippia alba : interactions and potential for the development of antithrombotics.

Citrus sinensis and Lippia alba are herbal medicines widely used in the form of tea (infusion, decoction), which ethanolic extracts have already shown great anticoagulant activity in vitro . For this reason, they seem to be excellent candidates for the development of new antithrombotics and also have the potential to interact with them. The aim of this study was to evaluate the activity of aqueous extracts in blood coagulation and platelet aggregation, in addition to analysing the micromolecular composition of these species. Thrombin generation test (TGT) by the Calibrated Automated Thrombogram method and Platelet Aggregation Test by turbidimetry were performed to evaluate the biological activities, while the chemical composition was qualitatively evaluated using high-performance liquid chromatography. Aqueous extracts were elaborated according to the folk use. All extracts were effective in reducing thrombin formation in TGT. Infusion of L. alba and infusion and decoction of C. sinensis at a concentration of 0.6 mg/ml significantly reduced platelet aggregation induced by ADP, and only the decoction of L. alba at the same concentration was able to significantly reduce collagen-induced platelet aggregation. The presence of phenylpropanoids and flavonoids in C. sinensis and L. alba extracts was verified. Furthermore, hesperidin was identified in C. sinensis through coinjection. C. sinensis and L. alba are rich in phenolics and demonstrated an in-vitro effect on important processes of haemostasis (blood coagulation, platelet agreggation), corroborating the potential of C. sinensis and L. alba for the development of antithrombotics and interact with them.

Evaluation of mesalazine polymeric conjugate in the treatment of actinic proctitis in rats.

PURPOSE: The present study aimed at testing a new formulation of mesalazine linked to chondroitin sulfate and its components alone in the treatment of actinic proctitis in rats. METHODS: Forty-seven female Wistar rats were submitted to pelvic radiation and divided into eight groups: control A, mesalazine A, chondroitin A, and conjugate A, gavage of the according substance two weeks after irradiation and sacrifice three weeks after oral treatment; control C, mesalazine C, chondroitin C, and conjugate C, sacrifice six weeks after oral treatment. The rectum was submitted to histological characterization for each of the findings: inflammatory infiltrate, epithelial degeneration, mucosal necrosis, and fibrosis. RESULTS: The inflammatory infiltrate was more intense in chondroitin A, mesalazine A, and conjugate C. The collagen deposition was less intense in chondroitin A, and mesalazine A, and more intense in control C. CONCLUSIONS: Mesalazine and chondroitin alone were efficacious in inducing a delayed inflammatory response, hence reducing the late fibrosis. The conjugate was able to induce an ever more delayed inflammatory response.

Topical Bimatoprost Insert for Primary Open-Angle Glaucoma and Ocular Hypertension Treatment - A Phase II Controlled Study.

INTRODUCTION: The most common treatment for Primary Open-Angle Glaucoma (POAG) is the daily use of eye drops. Sustained-release drug delivery systems have been developed to improve patient adherence by achieving prolonged therapeutic drug concentrations in ocular target tissues while limiting systemic exposure. The purpose of this study is to compare the efficacy and safety of bimatoprost inserts with bimatoprost eye drops in patients with POAG and Ocular Hypertension (OH). METHODS: We include OH and POAG patients aged between 40 and 75 years-old. Both OH and POAG patients had intraocular pressure (IOP) greater than 21 and ≤30 mmHg at 9:00 am without glaucoma medication and normal biomicroscopy. Five normal patients with IOP≤14 mmHg constitute the control group. A chitosan-based insert of bimatoprost was placed at the upper conjunctival fornix of the right eye. In the left eye, patients used one drop of LumiganTM daily at 10:00 pm. For statistical analysis, a two-way analysis of variance (ANOVA), Student t-test, and paired t-test is used. RESULTS: Sixteen POAG and 13 OH patients with a mean age of 61 years were assessed. In both eyes, IOP reduction was similar during three weeks of follow-up (19.5±2.2 mmHg and 16.9±3.1 mmHg), insert, and eye drop, respectively; P=0.165). The percentage of IOP reduction in the third week was 30% for insert and 35% for eye drops (P=0.165). No intolerance or discomfort with the insert was reported. Among the research participants, 58% preferred the use of the insert while 25% preferred eye drops, and 17% reported no preference. CONCLUSION: Bimatoprost-loaded inserts showed similar efficacy to daily bimatoprost eye drops during three weeks of follow up, without major side effects. This might suggest a possible change in the daily therapeutic regimen for the treatment of POAG and OH.

Evaluation of mesalazine polymeric conjugate in the treatment of actinic proctitis in rats

ABSTRACT Purpose: The present study aimed at testing a new formulation of mesalazine linked to chondroitin sulfate and its components alone in the treatment of actinic proctitis in rats. Methods: Forty-seven female Wistar rats were submitted to pelvic radiation and divided into eight groups: control A, mesalazine A, chondroitin A, and conjugate A, gavage of the according substance two weeks after irradiation and sacrifice three weeks after oral treatment; control C, mesalazine C, chondroitin C, and conjugate C, sacrifice six weeks after oral treatment. The rectum was submitted to histological characterization for each of the findings: inflammatory infiltrate, epithelial degeneration, mucosal necrosis, and fibrosis. Results: The inflammatory infiltrate was more intense in chondroitin A, mesalazine A, and conjugate C. The collagen deposition was less intense in chondroitin A, and mesalazine A, and more intense in control C. Conclusions: Mesalazine and chondroitin alone were efficacious in inducing a delayed inflammatory response, hence reducing the late fibrosis. The conjugate was able to induce an ever more delayed inflammatory response.

Efficacy of novel antiseptic product containing essential oil of Lippia origanoides to reduce intramammary infections in cows.

BACKGROUND AND AIM: The use of antimicrobials in the control of mastitis is of concern in public health due to their inefficiency in targeting microorganisms. Studies with medicinal plants have risen as an alternative to the use of conventional products. The objective of this study was to evaluate the efficacy of an experimental disinfectant based on the essential oil (EO) from Lippia origanoides in preventing the development of new intramammary infections (IMI) in Holstein cows. MATERIALS AND METHODS: The conventional protocol of pre- and post-milking was used and the control (Conventional treatment [CNV]) and experimental (Experimental treatment [PEX]) products containing EO at 120 µL/mL were applied by immersion. Individual milk samples were analyzed using sheep blood agar methodologies and biochemical tests. The efficiency of the treatment was defined by the presence or absence of Staphylococcus aureus, coagulase-negative Staphylococcus, and Streptococcus spp. RESULTS: There were no clinical and subclinical mastitis cases, no lesions in the mucosal of teats, nor dirt score between groups in this study. Both treatments did not influence the occurrence of IMI. CONCLUSION: The results revealed that PEX acts efficiently against microorganisms compared to the disinfection by the conventional product demonstrating the efficacy of the alternative product on the prevention of new IMIs in dairy cows.

Prevention of bone infection after open fracture using a chitosan with ciprofloxacin implant in animal model.

PURPOSE: To evaluate different concentrations of ciprofloxacin to prevent infection after open fracture contaminated with S. aureus in rats using absorbable local delivery system. METHODS: Fifty-two Wistar rats were assigned to six groups. After 4 weeks, all animals underwent 99mTc-ceftizoxima scintigraphy evaluation, callus formation measurement and histological analysis. ANOVA, t-Student and Kruskal Wallis were used for quantitative variables statistical analysis, whereas qui square and exact Fisher were used for qualitative variables. RESULTS: Treatment using 25% and 50% of ciprofloxacin incorporated at the fracture fixation device were effective in preventing bone infection compared to control group (p<0.05). Chitosan were not effective in preventing bone infection when used alone compared to control group (p>0.05). Histological findings demonstrated bone-healing delay with 50% of ciprofloxacin. No difference in callus formation were observed (p>0.05). CONCLUSION: Local delivery treatment for contaminated open fracture using chitosan with ciprofloxacin is effective above 25%.

Development and validation of analytical method by HPLC-DAD for determination of vasodilator active in pharmaceutical ophthalmic forms

The substance 4-Aminobenzamidine dihydrochloride (4-AD) is one of the degradation products of diminazene aceturate and has demonstrated antiglaucomatous potential. Glaucoma is the second leading cause of blindness worldwide; thus, new therapeutic alternatives must be studied, for example, the molecule 4-AD vehiculated into polymeric inserts for prolonged release. The present work aims to develop and validate an analytical method to quantify 4-AD in pharmaceutical ophthalmic forms. A HPLC was used with UV-Vis detector, at 290 ƞm and ACE® C18 column (125 × 4.6 mm, 5 µm), in which the mobile phase consists of phosphate buffer (pH 7.4) and triethylamine (30 mmol/L), under an isocratic flow of 1.0 mL/min. The retention time of 3.2 minutes was observed. The method was developed and validated in accordance with ANVISA recommendations and ICH guides. The linearity range was established between the concentrations 5 and 25 µg/mL (correlation coefficient r = 0.993). The accuracy, repeatability, and intermediate precision tests obtained a relative standard deviation less than or equal to 5%. In addition, the method was considered selective, exact. and robust, with pH being its critical factor. Therefore, the HPLC analysis method is robust and can be used to quantify 4-AD in pharmaceutical forms for ocular application.(AU)

Prevention of bone infection after open fracture using a chitosan with ciprofloxacin implant in animal model

Abstract Purpose To evaluate different concentrations of ciprofloxacin to prevent infection after open fracture contaminated with S. aureus in rats using absorbable local delivery system. Methods Fifty-two Wistar rats were assigned to six groups. After 4 weeks, all animals underwent 99mTc-ceftizoxima scintigraphy evaluation, callus formation measurement and histological analysis. ANOVA, t-Student and Kruskal Wallis were used for quantitative variables statistical analysis, whereas qui square and exact Fisher were used for qualitative variables. Results Treatment using 25% and 50% of ciprofloxacin incorporated at the fracture fixation device were effective in preventing bone infection compared to control group (p<0.05). Chitosan were not effective in preventing bone infection when used alone compared to control group (p>0.05). Histological findings demonstrated bone-healing delay with 50% of ciprofloxacin. No difference in callus formation were observed (p>0.05). Conclusion Local delivery treatment for contaminated open fracture using chitosan with ciprofloxacin is effective above 25%.

Maltodextrin and Gum Arabic-Based Microencapsulation Methods for Anthocyanin Preservation in Juçara Palm (Euterpe edulis Martius) Fruit Pulp.

The juçara fruits (Euterpe edulis Martius), native to the Atlantic Forest, are rich in anthocyanins. To preserve the anthocyanins in juçara fruit pulp, this study aimed to evaluate the effectiveness of microencapsulation by spray drying and freeze drying with maltodextrin (dextrose equivalent 16.5 to 19.5) and gum arabic in different proportions. The obtained microparticles were characterized by quantifying the total polyphenol and anthocyanin contents, by performing differential scanning calorimetry, thermogravimetry, and infrared spectroscopy and by using scanning electron microscopy to analyze the morphology of the particles. The total amount of polyphenols in the fruit pulp was 750 ± 16.7 mg GAE/100 g of the freeze-dried sample. The total anthocyanins in the fruit pulp was 181.25 ± 5.36 (mg/100 g). The microparticles were formed by employing maltodextrin and gum arabic in a 1:1 proportion as the polymeric matrix; the mixtures of pulp and polymeric matrix were prepared in proportions of 2:3 and 2:1, preserving up to 83.69% of the anthocyanin content. Lyophilization of the 2:1 mixture resulted in an anthocyanin content of 116.89 ± 4.43 (mg/100 g), whereas lyophilization of the 2:3 mixture resulted in 151.68 ± 1.39 (mg/100 g) anthocyanin content, which did not differ from the value obtained by spray drying the 2:3 mixture (150.76 ± 5.79 (mg/100 g)). Thermal analyses showed that the microparticles obtained by freeze drying at a ratio of 2:3 presented greater resistance to degradation with increasing temperature. The incorporation of the pulp in the polymeric matrix was demonstrated by IR analyses. Microparticles obtained by freeze drying showed the formation of various-sized flakes, whereas those obtained by spray drying were spherical in shape. Microencapsulation is a possible alternative for improving the stability of the anthocyanins in this fruit.

A rapid simultaneous determination of methylxanthines and proanthocyanidins in Brazilian guaraná (Paullinia cupana Kunth.).

Paullinia cupana is a plant native to Brazil that is widely used in traditional medicine as a physical and mental stimulant. It is also used worldwide to produce soft drinks. A method for the simultaneous quantitation of seven markers in guaraná by HPLC-PDA was developed, and extraction methods for the determination of methylxanthines and tannins were investigated. Quantified substances were theobromine, theophylline, caffeine, catechin, epicatechin, procyanidins A2 and B2. Results confirmed the satisfactory selectivity and linearity (r2≥0.99) within the mass ranges. Repeatability (RSD≤2.80%), intermediate precision (RSD≤4.47%), accuracy (recoveries from 90.59%-104.67%), and robustness were demonstrated. Extract 1 presented the contents: 0.0177% (±1.02%) for theobromine, 0.0131% (±1.14%) for theophylline, 2.9429% (±1.27%) for caffeine, 0.4563% (±1.02%) for catechin, 0.5515% (±1.05%) for epicatechin, 0.0607% (±2.80%) for A2 and 0.1035% (±1.39%) for B2. The method for simultaneous quantitation of seven chemical markers in guaraná proved to be reliable using a simple and convenient HPLC setup.

In vivo efficacy of a biotherapic and eugenol formulation against Rhipicephalus microplus.

The control of Rhipicephalus microplus is essential to prevent cattle discomfort and economic losses. However, increased resistance and acaricides inefficiency lead producers to adopt strategies that could result in the accumulation of chemical residues in meat and milk with possibilities of poisoning in animals and people. This scenario demonstrates the necessity of research into the identification of novel, effective and environmentally safe therapeutic options for cattle tick control. The objectives of this study were to develop and assess the efficacy of R. microplus biotherapic and of 5% eugenol for the control of R. microplus in artificially infested calves. Eighteen male 6-month-old Holstein calves were divided into three groups of six animals. In Group 1, the animals did not receive medication (control group); in Group 2, the animals received 1 mL of R. microplus biotherapic at dilution 6CH (centesimal Hahnemannian), orally administered twice daily. And in Group 3, they received a single application of eugenol 5% in the pour-on formulation. The median efficacy for biotherapy and eugenol 5% was respectively 10.13 and 13.97%; however, upon analyzing reproductive efficiency, it is noteworthy that the biotherapic had 45.86% efficiency and was superior to the action of eugenol (12.03%) after 37 days of treatment. The ultrastructural study provided information about the effects of R. microplus biotherapic on the ovaries of engorged females and showed disorganization in the deposition of the oocyte exochorion. The results suggest hatchability inhibition of larvae, interference in R. microplus reproduction and future possibilities for eco-friendly control of R. microplus with biotherapic 6CH.

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

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

New delivery systems for amphotericin B applied to the improvement of leishmaniasis treatment.

Leishmaniasis is one of the six major tropical diseases targeted by the World Health Organization. It is a life-threatening disease of medical, social and economic importance in endemic areas. No vaccine is yet available for human use, and chemotherapy presents several problems. Pentavalent antimonials have been the drugs of choice to treat the disease for more than six decades; however, they exhibit high toxicity and are not indicated for children, for pregnant or breastfeeding women or for chronically ill patients. Amphotericin B (AmpB) is a second-line drug, and although it has been increasingly used to treat visceral leishmaniasis (VL), its clinical use has been hampered due to its high toxicity. This review focuses on the development and in vivo usage of new delivery systems for AmpB that aim to decrease its toxicity without altering its therapeutic efficacy. These new formulations, when adjusted with regard to their production costs, may be considered new drug delivery systems that promise to improve the treatment of leishmaniasis, by reducing the side effects and the number of doses while permitting a satisfactory cost-benefit ratio.

New delivery systems for amphotericin B applied to the improvement of leishmaniasis treatment

Leishmaniasis is one of the six major tropical diseases targeted by the World Health Organization. It is a life-threatening disease of medical, social and economic importance in endemic areas. No vaccine is yet available for human use, and chemotherapy presents several problems. Pentavalent antimonials have been the drugs of choice to treat the disease for more than six decades; however, they exhibit high toxicity and are not indicated for children, for pregnant or breastfeeding women or for chronically ill patients. Amphotericin B (AmpB) is a second-line drug, and although it has been increasingly used to treat visceral leishmaniasis (VL), its clinical use has been hampered due to its high toxicity. This review focuses on the development and in vivo usage of new delivery systems for AmpB that aim to decrease its toxicity without altering its therapeutic efficacy. These new formulations, when adjusted with regard to their production costs, may be considered new drug delivery systems that promise to improve the treatment of leishmaniasis, by reducing the side effects and the number of doses while permitting a satisfactory cost-benefit ratio.

Bimatoprost-loaded ocular inserts as sustained release drug delivery systems for glaucoma treatment: in vitro and in vivo evaluation.

The purpose of the present study was to develop and assess a novel sustained-release drug delivery system of Bimatoprost (BIM). Chitosan polymeric inserts were prepared using the solvent casting method and characterized by swelling studies, infrared spectroscopy, differential scanning calorimetry, drug content, scanning electron microscopy and in vitro drug release. Biodistribution of 99mTc-BIM eye drops and 99mTc-BIM-loaded inserts, after ocular administration in Wistar rats, was accessed by ex vivo radiation counting. The inserts were evaluated for their therapeutic efficacy in glaucomatous Wistar rats. Glaucoma was induced by weekly intracameral injection of hyaluronic acid. BIM-loaded inserts (equivalent to 9.0 µg BIM) were administered once into conjunctival sac, after ocular hypertension confirmation. BIM eye drop was topically instilled in a second group of glaucomatous rats for 15 days days, while placebo inserts were administered once in a third group. An untreated glaucomatous group was used as control. Intraocular pressure (IOP) was monitored for four consecutive weeks after treatment began. At the end of the experiment, retinal ganglion cells and optic nerve head cupping were evaluated in the histological eye sections. Characterization results revealed that the drug physically interacted, but did not chemically react with the polymeric matrix. Inserts sustainedly released BIM in vitro during 8 hours. Biodistribution studies showed that the amount of 99mTc-BIM that remained in the eye was significantly lower after eye drop instillation than after chitosan insert implantation. BIM-loaded inserts lowered IOP for 4 weeks, after one application, while IOP values remained significantly high for the placebo and untreated groups. Eye drops were only effective during the daily treatment period. IOP results were reflected in RGC counting and optic nerve head cupping damage. BIM-loaded inserts provided sustained release of BIM and seem to be a promising system for glaucoma management.