Population Pharmacokinetic of the Diterpenes ent-Polyalthic Acid and Dihydro-ent-Agathic Acid from Copaifera Duckei Oil Resin in Rats.

Copaifera duckei oleoresin is a plant product extensively used by the Brazilian population for multiple purposes, such as medicinal and cosmetic. Despite its ethnopharmacological relevance, there is no pharmacokinetic data on this important medicinal plant. Due to this, we determined the pharmacokinetic profile of the major nonvolatile compounds of C. duckei oleoresin. The diterpenes ent-polyalthic acid and dihydro-ent-agathic acid correspond to approximately 40% of the total oleoresin. Quantification was performed using LC-MS/MS, and the validated analytical method showed to be precise, accurate, robust, reliable, and linear between 0.57 and 114.74 µg/mL plasma and 0.09 to 18.85 µg/mL plasma, respectively, for ent-polyalthic acid and dihydro-ent-agathic acid, making it suitable for application in preclinical pharmacokinetic studies. Wistar rats received a single 200 mg/kg oral dose (gavage) of C. duckei oleoresin, and blood was collected from their caudal vein through 48 h. Population pharmacokinetics analysis of ent-polyalthic and dihydro-ent-agathic acids in rats was evaluated using nonlinear mixed-effects modeling conducted in NONMEN software. The pharmacokinetic parameters of ent-polyalthic acid were absorption constant rate = 0.47 h-1, central and peripheral apparent volume of distribution = 0.04 L and 2.48 L, respectively, apparent clearance = 0.15 L/h, and elimination half-life = 11.60 h. For dihydro-ent-agathic acid, absorption constant rate = 0.28 h-1, central and peripheral apparent volume of distribution = 0.01 L and 0.18 L, respectively, apparent clearance = 0.04 L/h, and elimination half-life = 3.49 h. The apparent clearance, central apparent volume of distribution, and peripheral apparent volume of distribution of ent-polyalthic acid were approximately 3.75, 4.00-, and 13.78-folds higher than those of dihydro-ent-agathic.

Copaifera spp. oleoresins impair Toxoplasma gondii infection in both human trophoblastic cells and human placental explants.

The combination of pyrimethamine and sulfadiazine is the standard care in cases of congenital toxoplasmosis. However, therapy with these drugs is associated with severe and sometimes life-threatening side effects. The investigation of phytotherapeutic alternatives to treat parasitic diseases without acute toxicity is essential for the advancement of current therapeutic practices. The present study investigates the antiparasitic effects of oleoresins from different species of Copaifera genus against T. gondii. Oleoresins from C. reticulata, C. duckei, C. paupera, and C. pubiflora were used to treat human trophoblastic cells (BeWo cells) and human villous explants infected with T. gondii. Our results demonstrated that oleoresins were able to reduce T. gondii intracellular proliferation, adhesion, and invasion. We observed an irreversible concentration-dependent antiparasitic action in infected BeWo cells, as well as parasite cell cycle arrest in the S/M phase. The oleoresins altered the host cell environment by modulation of ROS, IL-6, and MIF production in BeWo cells. Also, Copaifera oleoresins reduced parasite replication and TNF-α release in villous explants. Anti-T. gondii effects triggered by the oleoresins are associated with immunomodulation of the host cells, as well as, direct action on parasites.

Anteroventral third ventricle (AV3V) lesion affects hypothalamic neuronal nitric oxide synthase (nNOS) expression following water deprivation.

Neuronal nitric oxide synthase (nNOS) has been reported to be up-regulated in the hypothalamic supraoptic nucleus (SON) during dehydration which in turn could increase nitric oxide (NO) production and consequently affect arginine vasopressin (AVP) secretion. The anteroventral third ventricle (AV3V) region has strong afferent connections with the SON. Herein we describe our analysis of the effects of an AV3V lesion on AVP secretion, and c-fos and nNOS expression in the SON following dehydration. Male Wistar rats had their AV3V region electrolytically lesioned or were sham operated. After 21 days they were submitted to dehydration or left as controls (euhydrated). Two days later, one group was anaesthetized, perfused and the brains were processed for Fos protein and nNOS immunohistochemistry (IHC). Another group was decapitated, the blood collected for hematocrit, osmolality, serum sodium and AVP plasma level analysis. The brains were removed for measurement of neurohypophyseal AVP content, and the SON was punched out and processed for nNOS detection by western blotting. The AV3V lesion reduced AVP plasma levels and c-fos expression in the SON following dehydration (P<0.05). Western blotting revealed an up-regulation of nNOS in the SON of control animals following dehydration, whereas such up-regulation was not observed in AV3V-lesioned rats (P<0.05). We conclude that the AV3V region plays a role in regulating the expression of nNOS in the SON of rats submitted to dehydration, and thus may affect the local nitric oxide production and the secretion of vasopressin.

Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats.

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100µg/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4, 6 or 24h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24h after surgery, and in both hypothalamic nuclei only at 6h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood.

Role of central NO-cGMP pathway in vasopressin and oxytocin gene expression during sepsis.

Sepsis induces massive production of inflammatory mediators, such as nitric oxide (NO), and causes neuroendocrine and cardiovascular alterations. This study investigates the involvement of the central NO-cGMP pathway in arginine vasopressin (AVP) and oxytocin (OXY) gene expression during sepsis induced by cecal ligation and puncture (CLP). Male Wistar rats received an i.c.v. injection of ODQ (0.25 µg/µL), a selective inhibitor of the heme site of soluble guanylate cyclase, or of 1% dymethilsulfoxide (DMSO), as vehicle. Thirty minutes after the injections, sepsis was induced by cecal ligation and puncture or the animals were sham operated. The ODQ pre-treatment did not alter the progressive NO increase observed after CLP. In the supraoptic nucleus (SON), this pretreatment increased the relative gene expression ratio of AVP and OXY in the initial phase of sepsis, but in the late phase, the gene expression of both hormones was reduced. In the paraventricular nucleus (PVN), soluble guanylate cyclase inhibition caused an even larger decrease in the relative gene expression ratio of AVP and OXY during sepsis. These results are indicative of a role of the NO-cGMP pathway in hormonal synthesis in the SON and PVN of the hypothalamus during polymicrobial sepsis.