Role of the serotonergic pathway in uterotonic activity of Ananas comosus (L.) Merr. - An in vitro and in vivo study.

BACKGROUND: Ananas comosus (L.) Merr. has been used as a traditional medicine in inducing abortion in many countries. Our previous in vitro experiments showed that the aqueous fraction (F4) of A. comosus extract stimulated the rat and human uterine contractions. PURPOSE: The aim of this study was to identify the bioactive compound and further investigate the molecular mechanism of F4 induced contraction and the in vivo uterotonic effect of F4. MATERIALS AND METHODS: Organ bath studies were employed to compare the stimulatory effect of F4 in non and late pregnant uterine tissue followed by isolation of protein from late pregnant uterine tissue for the western blot analysis. The PhysioTel transmitter was implanted in pregnant SD rats to measure the changes in intrauterine pressure (IUP). Analyses of the crude extract and active principle in F4 was performed using LC-HRMS. RESULTS: Ripe F4 in a similar manner as serotonin produced a greater stimulatory response in late pregnant than non-pregnant uterine tissue without significant change in potency; ripe F4 also increased ERK phosphorylation which eventually led to a significant increase of the final product, MMP-13. In pregnant rats (E18), oral ripe F4 (1.5 g.100 g-1 body weight) and ergometrine (1 mg) did not stimulate the uterine contraction probably due to the low level of estradiol and as a consequence low 5-HT receptors at the time of administration. In contrast, in postpartum rats, oral administration of F4 and ergometrine produced a significant increase in maximal IUP to 4.3 and 4.9 folds of basal IUP respectively. Contrary to the folklore use, unripe F4 did not stimulate the uterine activity during pregnancy and postpartum. Bioassay guided fractionation identified serotonin as a major bioactive compound in ripe F4. CONCLUSIONS: Our data clearly indicate that the uterotonic effect of ripe F4 is mediated via the serotonergic pathway and suggest that serotonin rich diet may increase the peripheral serotonin and implicate in diverse physiological functions, including uterine motility.

Potent tocolytic activity of ethyl acetate fraction of Ananas comosus on rat and human uteri.

The aim of this study was to investigate the tocolytic properties of Ananas comosus extract in rat and human uterine tissue in vitro and in the rat in vivo. Organ bath technique was employed to perform functional studies in vitro. The PhysioTel transmitter was implanted in SD rats to measure the changes in intrauterine pressure (IUP) in vivo. Analyses of F2 was performed using LC-HRMS. F2 produced a non-selective inhibitory response on oxytocin, prostaglandin F2α, acetylcholine and KCl. The inhibitory activity of F2 on oxytocin-induced contraction was not attenuated by propranolol, TEA, glibenclamide and indomethacin. Nω-Nitro-L-arginine, a nitric oxide synthase inhibitor, suppressed the maximal tocolytic activity of F2 by 25%. DIDS, an inhibitor of chloride channels, appeared to suppress the relaxant effect of F2. F2 suppressed the oxytocin-induced contraction in Ca2+ free solution. The in vivo tocolytic activity of F2 and ritodrine were observed in non-pregnant rats during the estrous stage by suppressing the frequency and amplitude of IUP peaks following intrauterine administration. Chemical analysis confirmed the involvement of citric acid in the tocolytic activity of F2. However, another less polar fraction is essential to accompany citric acid to produce such potent inhibitory response of F2. It is likely that F2 exerted tocolytic activity by multiple mechanisms, including antagonizing L-type Ca2+ channels, interfering with the intracellular Ca2+ release mechanism and releasing nitric oxide. F2 would be a promising candidate to develop as a tocolytic agent.

Endothelial colony forming cells from human umbilical cord blood improved severe erectile dysfunction in obese type II diabetic rats.

AIM: To investigate the effect of intracavernous injection of human umbilical cord blood derived endothelial colony forming cells (HUCB ECFCs) on erectile dysfunction (ED) in Zucker Diabetic Fatty (ZDF) rat model. METHODS: Erectile function was assessed by cavernous nerve electrostimulation in ZDF rats aged 20-28 weeks. Following confirmation of severe ED at the age of 28 weeks, 21 ZDF rats were randomly assigned to three experimental groups: 1 million ECFCs, 2 million ECFCs, and phosphate buffered saline (PBS). Four weeks after intracavernous injection, the efficacy of ECFCs was quantified by intracavernous pressure (ICP) measurement, Masson's trichrome staining, immunohistologic and immunoblot analyses and TUNEL assay. KEY FINDINGS: Intracavernous ECFC administration improved ICP in a dose-dependent manner in comparison to the age-matched PBS group. Functional improvement in ICP was accompanied by a significant restoration of the cavernosal endothelial and smooth muscle cell content and cavernosal nerve function. The percentage eNOS and nNOS positive cavernosal cells, and their respective protein expression levels and nNOS positive cells in the dorsal penile nerve in 2 million ECFCs treated groups were significantly higher than the PBS group. TUNEL stain quantification showed a significant decrease in cavernosal apoptosis following ECFC treatment. SIGNIFICANCE: The results are expected to provide a scientific basis to further study the clinical application of HUCB ECFCs in ameliorating ED in human. CONCLUSIONS: HUCB ECFCs significantly improved severe ED in ZDF rats through improvement of the nerve and endothelium function and restoration of smooth muscle in the cavernosum by overcoming the cavernosal apoptosis.

Investigation of uterotonic properties of Ananas comosus extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: In folklore medicine Ananas comosus (pineapple) is reputed to act as an abortifacient and in expectant women as a means of inducing labor. Several reports have claimed abortifacient property of A. comosus fruit (ripe or unripe). Ripe fruit has been used orally as traditional medicine in inducing abortion in Kerala state of India while the juice of unripe fruit was used for abortion in Bangladesh. However, scientific evidence supporting the efficacy of pineapple extracts in inducing uterine contractions is clearly lacking. AIM OF THE STUDY: This study investigated the pharmacological effects of different fractions of pineapple extract with a range of maturities to identify the most potent uterotonic fraction. MATERIALS AND METHODS: The ethanolic crude extracts of pineapple (edible part) were prepared and fractionated through a series of liquid-liquid partitions. Fractions were separately tested on isolated uterine muscle from pregnant SD rats and human pregnant myometrium, which were cut into strips along the longitudinal axis of uterus. The strips were mounted vertically in organ baths (37°C) and exposed to cumulative addition of fractions (0.1-10mgml-1), serotonin (0.05-5µM) and different inhibitors to delineate the mechanism of action of the active ingredients of the extract. RESULTS: Aqueous fraction (F4) possesses uterine stimulant property which was blocked by verapamil but unaffected by indomethacin, prazosin and atosiban. Notably, ketanserin (10µM) diminished the maximal contractile response induced by both F4 and 5HT by 74.3% and 92.1% respectively. CONCLUSIONS: These results may indicate the presence of 5HT or 5HT-like compound(s) and serotonergic pathways may contribute to the uterotonic activity of pineapple extract.