Estudo comparativo dos perfis fitoquímicos e citotóxicos do extrato alcoólico de folhas de graviola (annona muricata) e fruta do conde (annona squamosa) / Comparative study of the phytochemical and cytotoxic profiles of the alcoholic extract of soursop (annona muricata) and custard apple (annona squamosa) leaves / Estudio comparativo de los perfiles fitoquímicos y citotóxicos del extracto alcohólico de hojas de guanábana (annona muricata) y del conde (annona squamosa)

Introdução: As plantas apresentam potencial medicinal devido aos metabólitos secundários e possuem compostos com alto potencial anti-inflamatório, com possibilidades terapêuticas. Das espécies vegetais de interesse destacamos e comparamos a graviola (Annona muricata) e a fruta do conde (Annona squamosa). Objetivo: Comparar o potencial do extrato alcoólico bruto de folhas da graviola e fruta do conde em ensaios fitoquímicos e citotóxicos. Método: Os extratos de folhas de graviola e fruta do conde foram obtidos por percolação, com o uso de 20g de folhas secas e trituradas com 100 ml de álcool de cereais. Na padronização dos extratos foram utilizadas análises de idenficação de componentes químicos, os taninos, flavonoides, saponinas e alcaloides, por meio de difrentes reações químicas e os antioxidantes foram analisados pela atividade do DPPH. A citotoxicidade das diferentes concentrações dos extratos também foram analisadas por hemólise e pelo teste da membrana corioalantide (CAM). Resultados: As análises fitoquímicas indicaram a presença de alcaloides, taninos, flavonoides e saponinas para ambos os extratos com alta capacidade antioxidante. Os testes de hemólise e CAM mostraram ausência de citotoxicidade na concentração de 2% e 4% para ambos os extratos e elevada citotoxicidade em 10% para o extrato de graviola. Conclusão: O extrato alcoólico das folhas da graviola e fruta do conde mostraram importante perfil antioxidante e com compostos anti-inflamatórios com pouca diferença entre os extratos o que estimula estudos futuros e continuidade para exploração anti-inflamatória.

Introduction: Plants have medicinal potential due to secondary metabolites and have compounds with high antiinflammatory potential, with therapeutic possibilities. Of the plant species of interest we highlight and compare the graviola (Annona Muricata) and the fruit of the count (Annona squamosa). Objective: To compare the potential of crude alcoholic extract of soursop and conde fruit leaves in phytochemical and cytotoxic assays. Method: The leaves extracts of soursop and fruit of the count were obtained by percolation, with the use of 20g of dry leaves and crushed with 100 ml of grain alcohol. In the standardization of the extracts were used idenfication analyzes of chemical components, tannins, flavonoids, saponins and alkaloids, through different chemical reactions and antioxidants were analyzed by the activity of DPPH. The cytotoxicity of the different concentrations of the extracts were also analyzed by hemolysis and by the corioalantide membrane test (CAM). Results: Phytochemical analysis indicated the presence of alkaloids, tannins, flavonoids and saponins for both extracts with high antioxidant capacity. The hemolysis and CAM tests showed absence of cytotoxicity at the concentration of 2% and 4% for both extracts and high cytotoxicity in 10% for graviola extract. Conclusion: The alcoholic extract of the leaves of soursop and fruit of the count showed important antioxidant profile and with anti-inflammatory compounds with little difference between the extracts which stimulates future studies and continuity for anti-inflammatory exploration.

Introducción: Las plantas tienen potencial medicinal debido a los metabolitos secundarios y poseen compuestos con alto potencial antiinflamatorio, además de ser utilizadas en tratamientos terapéuticos. De las especies vegetales de interés, destacamos y comparamos la guanábana (Annona muricata) y la fruta del conde (Annona squamosa). Objetivo: Comparar el potencial del extracto alcohólico crudo de hojas de guanábana y chirimoya en ensayos fitoquímicos y citotóxicos. Método: El extracto de hojas de guanábana y fruta del conde se obtuvo por percolación, utilizando 20g de hojas secas y trituradas con 100 ml de alcohol de grano. En la estandarización de los extractos se utilizaron análisis para identificar componentes químicos, taninos, flavonoides, saponinas y alcaloides, a través de diferentes reacciones químicas y los antioxidantes fueron analizados por la actividad de DPPH. También se analizó la citotoxicidad de diferentes concentraciones de extractos por hemólisis y por la prueba de membrana de corioalantida (CAM). Resultados: Los análisis fitoquímicos indicaron la presencia de alcaloides, taninos y flavonoides y saponinas para ambos extractos con alta capacidad antioxidante. Las pruebas de hemólisis y CAM mostraron ausencia de citotoxicidad a una concentración del 4% y alta citotoxicidad a partir del 10% también para ambos extractos. Conclusión: El extracto alcohólico de hojas de guanábana y chirimoya mostró importantes perfiles antioxidantes y con compuestos antiinflamatorios, lo que estimula futuros estudios y continuidad para la exploración antiinflamatoria.

Inflammation, infection and preterm birth.

Preterm birth is the leading cause of perinatal morbidity and mortality. Pathological processes that have been linked with preterm birth infection and / or intrauterine inflammation are most frequently found associated with their induction. Studies in animal models and human research showed prior infections to the induction of labor, the anteriority of infection over labor induction, and the existence of a subclinical latency phase between these two phenomena. The ascending route from the vagina and the cervix is preponderant but also microorganisms may access the amniotic cavity and the fetus by other pathways. During inflammation associated to infection, Prostaglandins are released simultaneously with Nitric oxide and their overproduction could be detrimental. Prostaglandins promote uterine contractions contributing to embryonic and fetal expulsion. Therefore aberrant activation of the inflammatory response may cause premature labor and this does not seem to depend on how the microoorganisms accessed the uterus.

Interaction between lysophosphatidic acid, prostaglandins and the endocannabinoid system during the window of implantation in the rat uterus.

Bioactive lipid molecules as lysophosphatidic acid (LPA), prostaglandins (PG) and endocannabinoids are important mediators of embryo implantation. Based on previous published data we became interested in studying the interaction between these three groups of lipid derivatives in the rat uterus during the window of implantation. Thus, we adopted a pharmacological approach in vitro using LPA, DGPP (a selective antagonist of LPA3, an LPA receptor), endocannabinoids' receptor selective antagonists (AM251 and AM630) and non selective (indomethacin) and selective (NS-398) inhibitors of cyclooxygenase-1 and 2 enzymes. Cyclooxygenase isoforms participate in prostaglandins' synthesis. The incubation of the uterus from rats pregnant on day 5 of gestation (implantation window) with LPA augmented the activity and the expression of fatty acid amide hydrolase, the main enzyme involved in the degradation of endocannabinoids in the rodent uteri, suggesting that LPA decreased endocannabinoids' levels during embryo implantation. It has been reported that high endocannabinoids are deleterious for implantation. Also, LPA increased PGE2 production and cyclooxygenase-2 expression. The incubation of LPA with indomethacin or NS-398 reversed the increment in PGE2 production, suggesting that cyclooxygenase-2 was the isoform involved in LPA effect. PGs are important mediators of decidualization and vascularization at the implantation sites. All these effects were mediated by LPA3, as the incubation with DGPP completely reversed LPA stimulatory actions. Besides, we also observed that endocannabinoids mediated the stimulatory effect of LPA on cyclooxygenase-2 derived PGE2 production, as the incubation of LPA with AM251 or AM630 completely reversed LPA effect. Also, LPA augmented via LPA3 decidualization and vascularization markers. Overall, the results presented here demonstrate the participation of LPA3 in the process of implantation through the interaction with other groups of lipid molecules, prostaglandins and endocannabinoids, which prepare the uterine milieu for embryo invasion during the window of implantation.

Cyclooxygenase-2 prostaglandins mediate anandamide-inhibitory action on nitric oxide synthase activity in the receptive rat uterus.

Anandamide, an endocannabinoid, prostaglandins derived from cyclooxygenase-2 and nitric oxide synthesized by nitric oxide synthase (NOS), are relevant mediators of embryo implantation. We adopted a pharmacological approach to investigate if anandamide modulated NOS activity in the receptive rat uterus and if prostaglandins mediated this effect. As we were interested in studying the changes that occur at the maternal side of the fetal-maternal interface, we worked with uteri obtained from pseudopregnant rats. Females were sacrificed on day 5 of pseudopregnancy, the day in which implantation would occur, and the uterus was obtained. Anandamide (2 ng/kg, i.p.) inhibited NOS activity (P<0.001) and increased the levels of prostaglandin E(2) (P<0.001) and prostaglandin F(2α) (P<0.01). These effects were mediated via cannabinoid receptor type 2, as the pre-treatment with SR144528 (10 mg/kg, i.p.), a selective cannabinoid receptor type 2 antagonist, completely reverted anandamide effect on NOS activity and prostaglandin levels. The pre-treatment with a non-selective cyclooxygenase inhibitor (indomethacin 2.5mg/kg, i.p.) or with selective cyclooxygenase-2 inhibitors (meloxicam 4 mg/kg, celecoxib 3mg/kg, i.p.) reverted anandamide inhibition on NOS, suggesting that prostaglandins are derived from cyclooxygenase-2 mediated anandamide effect. Thus, anandamide levels seemed to modulate NOS activity, fundamental for implantation, via cannabinoid receptor type 2 receptors, in the receptive uterus. This modulation depends on the production of cyclooxygenase-2 derivatives. These data establish cannabinoid receptors and cyclooxygenase enzymes as an interesting target for the treatment of implantation deficiencies.

Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines.

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 µM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 µM L-NAME, a NO synthase inhibitor, or 30 µg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.

The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst.

Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1) h(-1)) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies.

Inflammatory agents involved in septic miscarriage.

Even though the understanding of the cause of early pregnancy loss due to chromosomal abnormalities has improved, there is a dearth of knowledge of the causes of loss in euploid conceptuses. Maternal infections are a cause of abort in humans, but the mechanisms are not clear, so we have developed a murine model to study the mechanism of septic abortion by inducing embryonic resorption (ER) with lipopolysaccharide (LPS). We demonstrated that augmented production of nitric oxide (NO) and prostaglandins (PG) is involved in ER, and that inhibitors of their synthesis could prevent ER. Also, we observed an increase in the oxidative damage, evidenced by nitration of tyrosine proteins, due to the peroxynitrite anion. Since an association between chronic marijuana smoking and early miscarriage has been shown in women, we studied the participation of anandamide (AEA), the principal endocannabinoid, on the mechanism of action of LPS. We showed that LPS-induced NO synthesis and tissue damage were mediated by AEA, and that this endotoxin inhibited AEA degradation and increased its synthesis. These results suggest that several inflammatory molecules participate in the mechanism of early pregnancy loss and that their modulation could be useful tools to prevent it.

The endocannabinoid system in bull sperm and bovine oviductal epithelium: role of anandamide in sperm-oviduct interaction.

Anandamide binds to cannabinoid receptors and plays several central and peripheral functions. The aim of this work was to study the possible role for this endocannabinoid in controlling sperm-oviduct interaction in mammals. We observed that bull sperm and bovine oviductal epithelial cells express cannabinoid receptors, CB1 and CB2, and fatty acid amide hydrolase, the enzyme that controls intracellular anandamide levels. A quantitative assay to determine whether anandamide was involved in bovine sperm-oviduct interaction was developed. R(+)-methanandamide, a non-hydrolysable anandamide analog, inhibited sperm binding to and induced sperm release from oviductal epithelia. Selective CB1 antagonists (SR141716A or AM251) completely blocked R(+)-methanandamide effects. However, SR144528, a selective CB2 antagonist, did not exert any effect, indicating that only CB1 was involved in R(+)-methanandamide effect. This effect was not caused by inhibition of the sperm progressive motility or by induction of the acrosome reaction. Overall, our findings indicate for the first time that the endocannabinoid system is present in bovine sperm and oviductal epithelium and that anandamide modulates the sperm-oviduct interaction, by inhibition of sperm binding and induction of sperm release from oviductal epithelial cells, probably by activating CB1 receptors.

Secretory and cytosolic phospholipase A2 activities and expression are regulated by oxytocin and estradiol during labor.

The release of arachidonic acid from membrane glycerophospholipids through the action of phospholipases (PLs) is the first step in the biosynthesis of prostaglandins (PGs). In reproductive tissues, the most important PLs are cytosolic PLA(2) (cPLA(2)) and types IIA and V of the secretory isoform (sPLA(2)). The aim of this work was to investigate the role of ovarian steroid hormones and oxytocin (OT) in the regulation of rat uterine PLA(2) activity and expression during pregnancy and labor. The activity of sPLA(2) increased near labor, whereas cPLA(2) activity augmented towards the end of gestation. The levels of sPLA(2) IIA and cPLA(2) mRNA showed an increase before labor (P<0.05, day 21), whereas sPLA(2) V mRNA was not regulated during pregnancy. The administration of atosiban (synthetic OT antagonist) together with tamoxifen (antagonist of estrogen receptors) was able to decrease cytosolic and secretory PLA(2) activities, diminish the expression of sPLA(2) IIA and cPLA(2), as well as decrease PGF(2 alpha) production before the onset of labor (P<0.01). The ovarian steroid did not affect PLA(2) during pregnancy. Collectively, these findings indicate that in the rat uterus, both 17beta-estradiol and OT could be regulating the activity and the expression of the secretory and the cytosolic isoforms of PLA(2), thus controlling PGF(2 alpha) synthesis prior to the onset of labor.

Different mechanisms lead to the angiogenic process induced by three adenocarcinoma cell lines.

Neoangiogenesis is essential for tumor and metastasis growth, but this complex process does not follow the same activation pathway, at least in tumor cell lines originated from different murine mammary adenocarcinomas. LMM3 cells were the most potent to stimulate new blood vessel formation. This response was significantly reduced by preincubating cells with indomethacin and NS-398, non-selective cyclooxygenase (COX) and COX-2 selective inhibitors, respectively. COX-1 and COX-2 isoenzymes were both highly expressed in LMM3 cells, and we observed that indomethacin was more effective than NS-398 to inhibit prostaglandin E2 (PGE2) synthesis. In addition, nitric oxide synthase (NOS) inhibitors, Nomega monomethyl L-arginine and aminoguanidine, also reduced LMM3-induced angiogenesis and nitric oxide (NO) synthesis as well. NOS2 > NOS3 proteins and arginase II isoform were detected in LMM3 cells by Western blot. The latter enzyme was also involved in the LMM3 neovascular response, since the arginase inhibitor, Nomega hydroxy L-arginine reduced the angiogenic cascade. On the other hand, parental LM3 cells were able to stimulate neovascularization via COX-1 and arginase products since only indomethacin and Nomega hydroxy L-arginine, which diminished PGE2 and urea synthesis, respectively, also reduced angiogenesis. In turn, LM2 cells angiogenic response could be due in fact to PGE2-induced VEGF liberation that stimulated neoangiogenesis at very low levels of NO.

IL-10 inhibits nitric oxide synthesis in murine uterus.

OBJECTIVES: Recent reports point to a role for the nitric oxide/nitric oxide synthase (NO/NOS) system in implantation. It has been suggested that inducible NOS expressed at peri-implantation would lead to enhanced NO production, which could promote the attachment of the blastocyst. Short-term administration of NO donors during the pre-implantation period reduced the pregnancy rate in a dose-dependent manner. Thus, it is thought that optimal levels of NO are critical for embryo implantation, so regulation of NOS must be crucial. Taking this into consideration, interleukin-10 (IL-10), synthesized and secreted by the embryo, could be modulating NOS during implantation. In this study we have investigated the in vitro effect of IL-10 on NOS in the uterus. METHODS: To determine the effect of IL-10, slices of uterus from estrogenized mice were pre-incubated for 60 min with different concentrations of IL-10 and NOS activity was measured. RESULTS: IL-10 (50 and 100 ng/ml in vitro) diminished NOS activity. The in vivo administration of lipopolysaccharide (LPS; 8 mg/kg) significantly increased the conversion of arginine into citrulline. This effect was abolished after 60 min of preincubation with IL-10 (100 ng/ml). The stimulatory effect of LPS and estrogen on NOS activity is exerted on the Ca-independent isoform and IL-10 in vitro abolished this increase. We observed that the uterus of pregnant mice on day 5 of gestation synthesized NO. This production was significantly inhibited by preincubation with IL-10 (100 ng/ml). CONCLUSIONS: This report demonstrates that IL-10 is capable of inhibiting NO synthesis in estrogenized, LPS-treated and pregnant rat uterus.