Ethyl Acetate Fraction from Trichilia catigua Confers Partial Neuroprotection in Components of the Enteric Innervation of the Jejunum in Diabetic Rats.

BACKGROUND/AIMS: Diabetes causes damage to the enteric nervous system. The enteric nervous system consists of neurons and enteric glial cells (EGCs). The present study evaluated the effects of an ethyl-acetate fraction (EAF) from Trichilia catigua (T. catigua; 200 mg/kg) on the total population of enteric neurons (HuC/D-immunoreactive [IR]) and EGCs (S100-IR and glial fibrillary acidic protein [GFAP]-IR) in the total preparation and jejunal mucosa in diabetic rats. METHODS: The animals were distributed into four groups: normoglycemic rats (N), diabetic rats (D), normoglycemic rats that received the EAF (NC), and diabetic rats that received the EAF (DC). The jejunum was processed for immunohistochemistry to evaluate HuC/D, S100, and GFAP immunoreactivity. The expression of S100 and GFAP proteins was also quantified by Western blot. RESULTS: The D group exhibited a decrease in the number of neurons and EGCs, an increase in the area of cell bodies, an increase in S100 protein expression, a decrease in GFAP protein expression, and a decrease in S100-IR and GFAP-IR EGCs in the jejunal mucosa. The DC group exhibited a decrease in the number of neurons and EGCs, a decrease in the area of cell bodies, a decrease in S100 and GFAP protein expression, and a decrease in S100-IR and GFAP-IR EGCs in the jejunal mucosa. The NC group exhibited maintenance of the number of neurons and EGCs, an increase in the area of cell bodies, and a decrease in S100 and GFAP protein expression. CONCLUSION: The EAF from T. catigua partially conferred protection against diabetic neuropathy in the enteric nervous system.

Anti-Diabetic Effects of the Ethyl-Acetate Fraction of Trichilia catigua in Streptozo-tocin-Induced Type 1 Diabetic Rats.

BACKGROUND/AIMS: Trichilia catigua A. Juss., known as "catuaba" in Brazil, has been popularly used as a tonic for fatigue, impotence and memory deficits. Previously, our group demonstrated that the ethyl-acetate fraction (EAF) of T. catigua has antioxidant and anti-inflammatory effects. The present study evaluated the anti-diabetic activity of EAF in type 1 diabetic rats. METHODS: Male Wistar rats were divided into four groups (N: non-diabetic group, D: type 1 diabetic group, NC: non-diabetic + EAF group and DC: type 1 diabetic + EAF group). The latter two groups were treated with 200 mg/kg EAF. Type 1 diabetes was induced by intravenous streptozotocin (STZ) injection (35 mg/kg). Starting two days after STZ injection, EAF was administered daily by gavage for 8 weeks. RESULTS: EAF attenuated body mass loss and reduced food and water intake. EAF improved hyperglycaemia and other biochemical parameters, such as alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Furthermore, the number of pancreatic ß-cells and the size of the islets had increased by ß-cell proliferation in the DC group. EAF promoted reduction in kidney tissue damage in STZ-induced diabetic rats by reduction of renal fibrosis. CONCLUSION: The present study showed that EAF improves glucose homeostasis and endocrine pancreas morphology and inhibits the development of diabetic nephropathy in STZ-induced diabetic rats.

Capillary Electrophoresis as Tool for Diastereomeric Separation in a Trichilia catigua Fraction.

INTRODUCTION: The tree Trichilia catigua, popularly known as "catuaba", shows several biological activities and has emerged as a potential source of new drugs. Considering that more than 10 species are known under the same popular name, regulatory agencies require more rigorous quality control of this medicinal plant. OBJECTIVE: To develop and validate a methodology using capillary electrophoresis (CE) with ultraviolet (UV) detection for analysing polyphenols in the ethyl-acetate fraction (EAF) of Trichilia catigua. METHODOLOGY: Different electrophoretic conditions (such as wavelength of UV detection, voltage, buffer concentration and pH, cyclodextrin type and concentration) were investigated. After optimisation, borate buffer 80 mmol/L at pH 8.80 with 2-hydroxypropyl-ß-cyclodextrin 10 mmol/L was selected as background electrolyte. A voltage reduction was used to improve the separation of a diastereomeric pair of cinchonains. RESULTS: The method proved to be simple, sensitive, accurate, linear, precise and reproducible. For the first time in natural products analysis, a voltage reduction and hydroxypropyl-ß-cyclodextrin were used to improve the separation of diastereomeric pairs. Until now, this is the only described methodology able to separate catechin, epicatechin, cinchonains Ia, Ib, IIa, and IIb from Trichilia catigua samples on the same run in less than 12 min. When compared to the high performance liquid chromatography with photo-diode array detection (HPLC-PDA) method previously developed by our research group, the CE method was more efficient, faster, less expensive and less polluting. CONCLUSION: Our results demonstrate that this method could be employed in a quality-control laboratory for the quantification of polyphenols in EAF of Trichilia catigua. Copyright © 2016 John Wiley & Sons, Ltd.

Development and characterization of multiple emulsions for controlled release of Trichilia catigua (Catuaba) extract.

Considering the antioxidant activity of the Trichilia catigua extract (TCE), the aim of the current study was to develop and characterize W/O/W multiple emulsions containing different vegetable oils as a platform to deliver a TCE. The extract displayed antioxidant activity (IC50) of 4.59 µg/mL and total phenol content (TPC) of 50.84%. Formulations were prepared by the phase-inversion emulsification method and analyzed for morphological appearance, pH, conductivity, droplet size and distribution, content of active, rheological properties, in vitro release, skin permeation, and stability. Formulations prepared with canola oil were selected and displayed regular morphology, mean diameter 2.77 µm (without TCE), 3.07 µm with 0.5% and 3.23 µm with 1.0% TCE. Rheometry (flow) showed pseudoplastic behavior with minimal thixotropy for both systems. TCE could be released from emulsions containing 1.0% and 0.5% TCE in a controlled manner for 16 and 23 h, respectively. The emulsions allowed good retention of TCE in the skin (stratum corneum, epidermis, and dermis). In a 180-d assessment of accelerated chemical stability, TPC was more reduced for the emulsions at 40 °C; other parameters remained stable. Multiple emulsions containing TCE were developed, exhibited good characteristics, and may be considered for future investigations as anti-aging formulations for the skin.

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine-treated rats.

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H(1)- and H(2)-receptors. Cimetidine is a H(2)-receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated. Adult male rats were treated with 100 mg kg(-1) of cimetidine for 50 days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate-resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) method and immunohistochemical reactions for detecting caspase-3 and RANKL were performed. The number of TRAP-positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL-positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP-positive osteoclasts with TUNEL-positive nuclei and caspase-3-immunolabeled osteoclasts were found. A significant reduction in the number of TRAP-positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL-positive cells was observed in CimG. The significant reduction in the number of osteoclasts may be due to cimetidine-induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis.

Development and validation studies for determination of phenylpropanoid-substituted flavan-3-ols in semipurified extract of Trichilia catigua by high-performance liquid chromatography with photodiode array detection.

The phenolic compounds are the main phytochemical constituents of the bark of Trichilia catigua and are commonly used for medicinal purposes. An HPLC method for the simultaneous quantification of phenolic compounds (procyanidin B2 (PB2), epicatechin (EPC), chinchonains Ia, Ib, IIa, IIb, catechin, and chrologenic acid) in T. catigua extract was developed and validated. A suitable chromatographic system was selected, which uses a gradient elution with methanol/ACN (75:25), and water both with 0.05% TFA, as mobile phase, column Luna, 280 nm, and flow 0.4 mL/min. Validation of the analytical method was based on the parameters: linearity, precision, LODs and LOQs, accuracy, robustness, and stability. The method showed linearity for PB2 and EPC, in the range 10-120 µg/mL with good correlation coefficients (>0.996). For precision, the repeatability ranged from 1.89 to 3.23%, and the values for accuracy for PB2 and EPC were 95 and 89%, respectively. The LODs and LOQs for PB2 were 1.36 and 4.12 µg/mL, and for EPC were 2.18 and 6.61 µg/mL, respectively. The method was robust under the conditions employed. The proposed method could be employed for quality assessment of T. catigua, as well as pharmaceutical products.

Antileishmanial activity of 5-methyl-2,2' : 5',2″-terthiophene isolated from Porophyllum ruderale is related to mitochondrial dysfunction in Leishmania amazonensis.

Recently, our group isolated and reported the antiproliferative activity in promastigotes and axenic amastigote forms of Leishmania amazonensis treated with 5-methyl-2,2':5',2″-terthiophene (compound A) and 5'-methyl-[5-(4-acetoxy-1-butynyl)]-2,2'-bi-thiophene (compound B) isolated from the aerial parts of Porophyllum ruderale. Here, we demonstrated that both compounds exhibited activity against intracellular amastigotes showing IC50 values of 37 and 51 µg/mL for compounds A and B, respectively. Both compounds showed low levels of toxicity for human cells, even at the highest concentrations (hemolytic index < 10 % at 500 µg/mL). Promastigotes treated with compound A showed an alteration in the mitochondrial membrane when observed by flow cytometry through labeling with rhodamine 123 and this was confirmed by transmission electron microscopy. Alterations on morphology (rounded cells) were observed by scanning electron microscopy in parasites treated with the compounds. Further studies should be performed employing compounds A and B for the development of new drugs for chemotherapy of leishmaniasis.

Effects of maternal exposure to extract of Trichilia catigua on antibody production in the offspring of Wistar rats.

It was evaluated the effects of maternal treatment with the Trichilia catigua (ExTc) crude extract on the antibodies' production by their offspring. Female rats received ExTc or saline from the first day of pregnancy until the twenty-first day after the birth of the pups, when the pups were weaned. All pups were inoculated with two doses of 50 µg of IgY diluted in aluminium hydroxide/PBS on days 26 and 40 of life. Antibody levels were analysed by ELISA. Our results show an increase in levels of IgG1 and IgG2a anti-IgY in female offspring of mothers treated with ExTc compared to female offspring of untreated mothers. Furthermore, ExTc treatment suppressed the production of IgG2a anti-IgY antibodies in males. The data show that maternal exposure to ExTc can modulate the production of antibodies in the offspring.

Treatment with Trichilia catigua ethyl-acetate fraction improves healing and reduces oxidative stress in TNBS-induced colitis in rats.

Beverages containing Trichilia catigua are commonly employed in folk medicine. T. catigua bark extracts possess antioxidant, anti-inflammatory, and bactericidal properties. These properties suggest T. catigua bark extracts as a potential treatment for inflammatory bowel diseases (IBD). Using the 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced model of colitis in rats we evaluated the effect of an ethyl-acetate fraction (EAF) of T. catigua (200 mg/kg) administered by daily oral gavage or intrarectally at different time points after TNBS challenge. TNBS treatment evoked severe colonic inflammation after 24 h that persisted for 7 days, characterized by weight loss, high levels of myeloperoxidase activity, histological and macroscopic damage, and elevated index of oxidative stress in the blood. T. catigua EAF treatment prevented the oxidative stress within 24 h and enhanced tissue recovery observed at day 7, returning histological and macroscopic damage levels to that of the control group. TNBS treatment led to loss of myenteric neurons after 28 days. T. catigua EAF was unable to prevent the neuronal loss. Oral delivery of T. catigua EAF was more effective than intrarectal administration of the extract. In conclusion, T. catigua EAF treatment normalized oxidative stress parameters in blood and reduced the degree of acute inflammation in TNBS colitis.

Ethyl-acetate fraction of Trichilia catigua restores long-term retrograde memory and reduces oxidative stress and inflammation after global cerebral ischemia in rats.

We originally reported that an ethyl-acetate fraction (EAF) of Trichilia catigua prevented the impairment of water maze learning and hippocampal neurodegeneration after transient global cerebral (TGCI) in mice. We extended that previous study by evaluating whether T. catigua (i) prevents the loss of long-term retrograde memory assessed in the aversive radial maze (AvRM), (ii) confers hippocampal and cortical neuroprotection, and (iii) mitigates oxidative stress and neuroinflammation in rats that are subjected to the four vessel occlusion (4-VO) model of TGCI. In the first experiment, naive rats were trained in the AvRM and then subjected to TGCI. The EAF was administered orally 30min before and 1h after TGCI, and administration continued once per day for 7days post-ischemia. In the second experiment, the EAF was administered 30min before and 1h after TGCI, and protein carbonylation and myeloperoxidase (MPO) activity were assayed 24h and 5days later, respectively. Retrograde memory performance was assessed 8, 15, and 21days post-ischemia. Ischemia caused persistent retrograde amnesia, and this effect was prevented by T. catigua. This memory protection (or preservation) persisted even after the treatment was discontinued, despite the absence of histological neuroprotection. Protein carbonyl group content and MPO activity increased around 43% and 100%, respectively, after TGCI, which were abolished by the EAF of T. catigua. The administration of EAF did not coincide with the days of memory testing. The data indicate that antioxidant and/or antiinflammatory actions in the early phase of ischemia/reperfusion contribute to the long-term antiamnesic effect of T. catigua.

Cimetidine Reduces Interleukin-6, Matrix Metalloproteinases-1 and -9 Immunoexpression in the Gingival Mucosa of Rat Molars With Induced Periodontal Disease.

BACKGROUND: Histamine seems to act, via H2 receptor, on inflammatory processes by stimulating interleukin (IL)-6 and matrix metalloproteinase (MMP) release. As cimetidine is an H2 receptor antagonist, the authors hypothesize that this antiulcer drug reduces IL-6, MMP-1, and MMP-9 immunoexpression in gingiva with induced periodontal disease (PD). To confirm a possible modulatory role of IL-6 on MMPs, the relationship between IL-6/MMP-1 and IL-6/MMP-9 immunoexpression was evaluated. METHODS: Forty-six male rats were distributed into the cimetidine group (CimG: received daily intraperitoneal injections of 100 mg/kg of body weight of cimetidine) or the saline group (SG). PD was induced by cotton ligature around the maxillary left first molars (PDSG and PDCimG). The right molars were used as controls (SG and CimG). After 7, 15, 30, and 50 days, maxillary fragments were processed for paraffin embedding or for transmission electron microscopy. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the alveolar process surface and number of IL-6, MMP-1, and MMP-9-immunolabeled cells in the gingival mucosa were quantified. Statistical analyses were performed (P ≤0.05). RESULTS: In PDSG and PDCimG, gingival mucosa exhibited few collagen fibers among numerous inflammatory cells. In PDCimG, the number of TRAP-positive osteoclasts and IL-6, MMP-1, and MMP-9-immunolabeled cells was significantly lower than in PDSG at all periods. A positive correlation between IL-6/MMP-1 and IL-6/MMP-9 was detected in PDSG and PDCimG. CONCLUSION: Cimetidine decreases bone loss through reduction of osteoclast number and induces reduction of IL-6, MMP-1, and MMP-9 immunoexpression, reinforcing the idea that the beneficial effect of cimetidine in PD may be due to reduction of IL-6 immunolabeling in the inflamed gingival mucosa.

In vitro Cytotoxicity and Anti-herpes Simplex Virus Type 1 Activity of Hydroethanolic Extract, Fractions, and Isolated Compounds from Stem Bark of Schinus terebinthifolius Raddi.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) is associated with orofacial infections and is transmitted by direct contact with infected secretions. Several efforts have been expended in the search for drugs to the treatment for herpes. Schinus terebinthifolius is used in several illnesses and among them, for the topical treatment of skin wounds, especially wounds of mucous membranes, whether infected or not. OBJECTIVE: To evaluate the cytotoxicity and anti-HSV-1 activity of the crude hydroethanolic extract (CHE) from the stem bark of S. terebinthifolius, as well as its fractions and isolated compounds. MATERIALS AND METHODS: The CHE was subjected to bioguided fractionation. The anti-HSV-1 activity and the cytotoxicity of the CHE, its fractions, and isolated compounds were evaluated in vitro by SRB method. A preliminar investigation of the action of CHE in the virus-host interaction was conducted by the same assay. RESULTS: CHE presented flavan-3-ols and showed anti-HSV-1 activity, better than its fractions and isolated compounds. The class of substances found in CHE can bind to proteins to form unstable complexes and enveloped viruses, as HSV-1 may be vulnerable to this action. Our results suggest that the CHE interfered with virion envelope structures, masking viral receptors that are necessary for adsorption or entry into host cells. CONCLUSION: The plant investigated exhibited potential for future development treatment against HSV-1, but further tests are necessary, especially to elucidate the mechanism of action of CHE, as well as preclinical and clinical studies to confirm its safety and efficacy. SUMMARY: Crude hydroethanolic extract (CHE) presents promising activity against herpes simplex virus type 1 (HSV 1), with selectivity index (SI) = 22.50CHE has flavan-3-ols in its composition, such as catechin and gallocatechinThe fractions and isolated compounds obtained from CHE by bioguided fractionation are less active than the CHE against HSV-1CHE interferes with viral entry process in the host cell and acts directly on the viral particle. Abbreviations used: HSV: Herpes simplex virus, CHE: Crude hydroethanolic extract, WF: Water fraction, AF: Ethyl-acetate fraction, MPLC: Medium-performance liquid chromatography, TLC: Thin-layer chromatography, NMR: Nuclear magnetic resonance, ESI-MS: Electrospray ionization mass spectrometry, SRB: Sulforhodamine B, CPE: Cytopathic effect, CC50: 50% cytotoxic concentration, EC50: 50% effective concentration, PBS: Phosphate-buffered saline.

Trichilia catigua ethyl-acetate fraction protects against cognitive impairments and hippocampal cell death induced by bilateral common carotid occlusion in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Trichilia catigua preparations have antinociceptive, antiinflammatory, and neuroprotective activity. Recently, a neuroprotective role for T. catigua was proposed using an in vitro model of ischemia-reperfusion in rat hippocampal slices. The aim of the present study was to evaluate the effects of an ethyl-acetate fraction (EAF) of T. catigua, which has potent antioxidant activity, in mice subjected to an in vivo model of cerebral ischemia. MATERIAL AND METHODS: Male Swiss mice were subject to the bilateral common carotid occlusion (BCCAO) model of cerebral ischemia. The animals were orally administered the T. catigua EAF (200, 400, or 800 mg/kg) 30 min before and once per day for 7 days after BCCAO. Histological and behavioral outcomes were assessed using Nissl staining and the Morris water maze test of cognition, respectively. RESULTS: Mice that were subjected to BCCAO exhibited cognitive impairments in the Morris water maze. The spatial cognitive deficits were counteracted by T. catigua EAF administration (200-800 mg/kg). The T. catigua EAF significantly increased the number of intact-appearing Nissl-stained cells in the hippocampus in BCCAO mice. CONCLUSIONS: These results show that the T. catigua EAF promoted functional recovery, decreased the delayed hippocampal cell loss, and mitigated the ongoing neurodegenerative processes induced by BCCAO in mice.

Antiviral Activity of Trichilia catigua Bark Extracts for Herpesvirus and Poliovirus.

Herpesvirus and poliovirus are responsible for important diseases in human and animal. Trichilia catigua a Brazilian native plant known as catiguá has several medicinal properties among them antimicrobial for bacteria and protozoa, however, no antiviral activity has been reported yet. This study evaluated the antiviral activity of the crude extract (CE) and aqueous and ethyl acetate fractions (AF, EAF) obtained from T. catigua in the replication of the Herpes simplex virus (HSV-1), bovine herpesvirus (BoHV-1) and poliovirus (PV-1). The cytotoxicity was analyzed by MTT assay and the antiviral effect was determined by the addition of extracts (0.25 to 100.0 µg/ml), before (-2h and -1h), during (Oh) and after (1h and 2h) the viral infection, by plaque reduction assay, in HEp-2 cell culture. The virucidal activity and inhibition of viral adsorption were also evaluated. In addition, the combination index (CI) with Acyclovir (ACV - reference drug) was determined for HSV-1. CE, AF and EAF showed a low toxicity (CC(50) >400 µg/ml) and low inhibitory concentration (IC50), ranging from 2.44-34.25 µg/ml for herpesvirus and 0.67 to 1.8 µg/ml for PV-1, associated with high selectivity index. The tested compounds showed high virucidal effect and high ability to inhibit viral adsorption, for all virus. The CI demonstrated a synergic effect (CI<1) for AF and EAF comparatively to acyclovir (ACV). Our study demonstrated that the extract and fractions of T. catigua is promising for future antiviral drug design with economically feasible production.

The exposure to Trichilia catigua (catuaba) crude extract impairs fertility of adult female rats but does not cause reproductive damage to male offspring.

ETHNOPHARMACOLOGICAL RELEVANCE: Trichilia catigua is broadly used in folk medicine due to its mental and physical tonic activities and stimulant effects. In animal models, its antidepressant-like effects have been associated with the dopaminergic (DA) system modulation, which has an important role on maternal behavior and male offspring reproductive development. AIM OF THE STUDY: Since little is known about the adverse effects of the exposure to T. catigua crude extract (CAT) in rats, specially regarding maternal homeostasis and offspring development, the aim of the present study was to evaluate whether CAT exposure may influence maternal toxicity parameters and behavior or disrupt male offspring physical and reproductive development. MATERIAL AND METHODS: Dams were treated daily (by gavage) with 400mg/kg of CAT or vehicle (control=CTR) throughout pregnancy and lactation. Fertility and maternal behavior tests were conducted in dams. Male offspring reproductive and behavioral parameters were analyzed. RESULTS: Dams exposed to CAT showed increased pre- and post-implantation losses rates when compared to CTR group. No significant changes regarding maternal behavior or male offspring parameters were observed. CONCLUSION: In conclusion, maternal exposure to CAT interfered with implantation during the initial phases of pregnancy but did not induce changes on maternal behavior or male offspring reproductive and behavioral parameters.

Cimetidine reduces alveolar bone loss in induced periodontitis in rat molars.

BACKGROUND: There is evidence that histamine released during inflammation plays a role in bone metabolism via the H2 receptor, stimulating bone resorption. The purpose of this study is to evaluate whether cimetidine, a histamine H2-receptor antagonist, interferes with the initiation and progression of induced periodontal disease in rat molars. METHODS: Forty male rats received 100 mg/kg body weight of cimetidine (cimetidine group [CimG]) or saline solution (sham group [SG]). Periodontal disease was induced in the maxillary left first molars (PDSG and PDCimG); maxillary right molars were used as non-ligature controls. After 7, 15, 30, and 50 days, maxillary fragments were embedded in paraffin. The sections were stained with Masson trichrome and hematoxylin and eosin and subjected to the tartrate-resistant acid phosphatase (TRAP) method. The distances between the cemento-enamel junction (CEJ) and alveolar process (AP) crest, as well as between the CEJ and junctional epithelium (JE) level, were measured; the number of inflammatory cells was computed. Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) immunohistochemistry was carried out, and the RANKL/OPG ratio was calculated. RESULTS: In PDSG and PDCimG, a significant increase (P ≤0.05) was observed in CEJ-AP and CEJ-JE distances. However, the increases in both distances were significantly less in PDCimG compared with PDSG at 15, 30, and 50 days. Numerous TRAP-positive osteoclasts were found in the PDSG and PDCimG. In PDCimG, the volume density of inflammatory cells and the RANKL/OPG ratio were significantly lower (P ≤0.05) than in PDSG. CONCLUSIONS: Cimetidine exerts a beneficial effect on periodontal disease in rats, decreasing the RANKL/OPG ratio in gingival connective tissue and reducing alveolar bone resorption.

Anxiolytic-like and proneurogenic effects of Trichilia catigua ethyl-acetate fraction in mice with cerebral ischemia

Abstract Trichilia catigua A. Juss., Meliaceae, known as "catuaba" in Brazil, has been popularly used as a tonic for fatigue, impotence and memory deficits. Previously, we have demonstrated that T. catigua ethyl-acetate fraction exerted antidepressive-like effects in mice. Affective-like symptoms are also well recognized outcome of cerebral ischemia in clinical and preclinical settings. Therefore, here we evaluated the effects of ethyl-acetate fraction on the emotional outcomes and its relation with hippocampal neurogenesis in ischemic mice. Male Swiss mice were subject to the bilateral common carotid occlusion during 20 min. The animals received ethyl-acetate fraction (400 mg/kg, orally) 30 min before and once per day during 7 days after reperfusion. Emotional outcomes were assessed using the open field test, elevated zero maze, and the tail suspension test. After the behavioral testing, the animals were sacrificed and their brains were processed to immunohistochemistry and Nissl staining. Ischemic mice exhibited anxiogenic-like behaviors in the elevated zero maze, hippocampal neurodegeneration and decreased hippocampal neurogenesis. The anxiogenic-like effect was counteracted by ethyl-acetate fraction administration. Furthermore, ethyl-acetate fraction restored the number of newborn neurons in the dentate gyrus of hippocampus of ischemic mice. In conclusion, T. catigua ethyl-acetate fraction promoted functional recovery and restored hippocampal neurogenesis in ischemic mice.

Simultaneous liquid chromatography- tandem mass spectrometry method to quantify epicatechin and procyanidin B2 in rat plasma after oral administration of Trichilia catigua (catuaba) extract and its application to a pharmacokinetic study

Abstract Trichilia catigua A. Juss., Meliaceae, known as catuaba in Brazil, is traditionally used for the treatment of stress, sexual impotence and memory deficits. To our knowledge, there is no analytical method described in literature for simultaneous quantification of catuaba extract marker substances in biological matrices. The aim of this study was to develop and validate a bioanalytical method by LC-MS/MS to quantify epicatechin and procyanidin B2 in rat plasma after administration of standardized extract of T. catigua. Chromatographic separation was achieved with a C18 column, methanol and 0.1% aqueous formic acid at a flow rate of 0.25 ml/min. Detection was performed using electrospray ionization in negative mode. The lower limits of quantification were 5 ng/ml and 12.5 ng/ml for procyanidin B2 and epicatechin, respectively. Intra- and inter-day assays variability were less than 15%. The extraction recovery was 104% for epicatechin and 74% for procyanidin B2 using one-step liquid-liquid extraction with ethyl acetate. Epicatechin and PB2 were detected in plasma up to 300 min after oral administration of 400 mg/kg of standardized extract of T. catigua in rats. This rapid and sensitive method for the analysis of the epicatechin and procyanidin B2 in rat plasma can be applied to pharmacokinetic studies.

Development of an analytical method using reversed-phase HPLC-PDA for a semipurified extract of Paullinia cupana var. sorbilis (guaraná).

The Neotropical plant 'guaraná' has been widely used in medicine, cosmetics, and industry because of its versatile biological activities. These effects are mainly attributed to the presence of polyphenols. An efficient, precise, and reliable method was developed for quantification of the polyphenols catechin and epicatechin in guaraná extract solution, using HPLC-PDA detection. The ideal conditions for the analysis of a semipurified extract of guaraná (EPA), using solutions of 0.05% TFA-water (phase A) and 0.05% TFA in acetonitrile:methanol (75:25, vv(-1)) (phase B) as mobile phases were established. Gradient reversed-phase chromatography was performed using a guard cartridge (C18, 4.6mm×20mm, 4µm) and column (C18, 250mm×4.6mm, 4µm), flow of 0.5mLmin(-1) and detection at 280nm. The main validation parameters of the method were also determined. The method was linear over a range of 18.75-300µgmL(-1) for catechin and epicatechin, with detection limits of 0.70 and 0.88µgmL(-1) and quantification limits of 2.13 and 2.67µgmL(-1), respectively. The method also showed consistent mean recoveries of 91.3±3.8%, 2.14 RSD and 93.4±3.1, 2.74 RSD of catechin and epicatechin respectively. The relative standard deviations were relatively low: intra-day (0.72% and 0.66% for catechin and epicatechin, respectively) and inter-day (0.93% and 0.75% for catechin and epicatechin, respectively). The semipurified extract showed catechin, epicatechin, and caffeine contents of 180.75, 278.87, and 300.87µgmg(-1), respectively. The results demonstrated the efficiency, precision, accuracy, and robustness of the proposed method. The solutions remained stable for a sufficient time (one week) to complete the analytical process.

Preliminary Assessment of the Chemical Stability of Dried Extracts from Guazuma ulmifolia Lam. (Sterculiaceae).

We report the results of a preliminary estimation of the stability of the dried extract from bark of Guazuma ulmifolia Lam. ("Mutamba"), with and without added colloidal silicon dioxide (CSD). The physical and chemical properties and the compatibility of CSD in the extract were evaluated for 21 days of storage under stress conditions of temperature (45 ± 2°C) and humidity (75 ± 5%). Thermogravimetry (TG) was supplemented using selective high-performance liquid chromatography (HPLC) for determination of stability of the characteristic constituents (chemical markers), namely, procyanidin B2 (PB2) and epicatechin (EP). The results showed that PB2 is an appropriate compound to be used as a chemical marker in the quality control of dried extracts of G. ulmifolia. The stress study showed that there was no significant difference between the two formulations. However, considering the TG data and the high temperatures involved, the results suggest that CSD increases the stability of the dried extract of G. ulmifolia.