Pharmacological insight into the anti-inflammatory activity of sesquiterpene lactones from Neurolaena lobata (L.) R.Br. ex Cass.

PURPOSE: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated sesquiterpene lactones raised our interest in further pharmacological studies. The present work aimed at providing a mechanistic insight into the anti-inflammatory activity of N. lobata and eight isolated sesquiterpene lactones, as well as a structure-activity relationship and in vivo anti-inflammatory data. METHODS: The effect of the extract and its compounds on the generation of pro-inflammatory proteins was assessed in vitro in endothelial and monocytic cells by enzyme-linked immunosorbent assay. Their potential to modulate the expression of inflammatory genes was further studied at the mRNA level. In vivo anti-inflammatory activity of the chemically characterized extract was evaluated using carrageenan-induced paw edema model in rats. RESULTS: The compounds and extract inhibited LPS- and TNF-α-induced upregulation of the pro-inflammatory molecules E-selectin and interleukin-8 in HUVECtert and THP-1 cells. LPS-induced elevation of mRNA encoding for E-selectin and interleukin-8 was also suppressed. Furthermore, the extract inhibited the development of acute inflammation in rats. CONCLUSIONS: Sesquiterpene lactones from N. lobata interfered with the induction of inflammatory cell adhesion molecules and chemokines in cells stimulated with bacterial products and cytokines. Structure-activity analysis revealed the importance of the double bond at C-4-C-5 and C-2-C-3 and the acetyl group at C-9 for the anti-inflammatory activity. The effect was confirmed in vivo, which raises further interest in the therapeutic potential of the compounds for the treatment of inflammatory diseases.

Metabolic effects of mulberry leaves: exploring potential benefits in type 2 diabetes and hyperuricemia.

The leaves of Morus alba L. have a long history in Traditional Chinese Medicine and also became valued by the ethnopharmacology of many other cultures. The worldwide known antidiabetic use of the drug has been suggested to arise from a complex combination effect of various constituents. Moreover, the drug is also a potential antihyperuricemic agent. Considering that type 2 diabetes and hyperuricemia are vice-versa in each other's important risk factors, the use of mulberry originated phytotherapeutics might provide an excellent option for the prevention and/or treatment of both conditions. Here we report a series of relevant in vitro and in vivo studies on the bioactivity of an extract of mulberry leaves and its fractions obtained by a stepwise gradient on silica gel. In vivo antihyperglycemic and antihyperuricemic activity, plasma antioxidant status, as well as in vitro glucose consumption by adipocytes in the presence or absence of insulin, xanthine oxidase inhibition, free radical scavenging activity, and inhibition of lipid peroxidation were tested. Known bioactive constituents of M. alba (chlorogenic acid, rutin, isoquercitrin, and loliolide) were identified and quantified from the HPLC-DAD fingerprint chromatograms. Iminosugar contents were investigated by MS/MS, 1-deoxynojirimycin was quantified, and amounts of 2-O-alpha-D-galactopyranosyl-1-deoxynojirimicin and fagomine were additionally estimated.

Physico-chemical comparison of betulinic acid, betulin and birch bark extract and in vitro investigation of their cytotoxic effects towards skin epidermoid carcinoma (A431), breast carcinoma (MCF7) and cervix adenocarcinoma (HeLa) cell lines.

Betulin and betulinic acid are pentacyclic triterpenes present in the bark of the birch tree and other vegetal sources. Quantitatively, in birch bark betulin is more significant than betulinic acid; therefore, birch can be a large and feasible source of raw material for betulin extraction. Betulin can be used as extracted or, after chemical modification, as a starting compound for its acid, betulinic acid, with both substances possessing various interesting pharmacological properties. The purpose of this study is to analyse the betulin and betulinic acid content of a birch tree bark extract, as well as its cytotoxic activity. The extraction was done using a Soxhlet extractor and chloroform/dichlormethane/methanol (1 : 1 : 1) as solvent. The betulin and betulinic acid content of the extract was estimated using standards of pure betulin and betulinic acid, by thermal analysis as opposed to pure substance (thermogravimetric and differential thermal analysis). The extract and the main compounds were also analysed by NMR. The results indicated a high amount of betulin in the final extract (up to 50%), and an important quantity of betulinic acid: over 3%. The cytotoxic activity indicated a high proliferation inhibition for the birch tree extract but was still comparable with betulinic acid and betulin.

The effects of adjuvant arthritis on the myometrial adrenergic functions in the nonpregnant and the late-pregnant rat.

The beneficial effects of pregnancy on the symptoms of inflammatory diseases are well documented. The modulation in the uterine functions in the presence of generalized inflammation, however, is much less characterized. The aim of the present study was to explore the modulatory action of adjuvant arthritis on the adrenergic functions of the uterus in nonpregnant and late pregnant rats. Adjuvant arthritis was induced by the subplantar injection of M. butyricum. Presynaptic functions were characterized by a superfusion technique and by registration of the contractions of isolated uterine rings elicited by electric field stimulation. The functions of the adrenoceptors were characterized by constructing concentration-response curves with agonists for both α- and ß-receptors. Where these curves differed significantly from the control, the expressions of these receptors at the mRNA level were additionally determined. Adjuvant arthritis substantially decreased the uptake and release of [(3)H]noradrenaline in myometrial samples from nonpregnant rats, but caused no change at term. The electrically induced contractions were decreased by inflammation in both gestational states. Arthritis resulted in decreased ß-adrenoceptor-mediated relaxation (in both the nonpregnant and the late-pregnant animals) and an increase in α-mediated contraction at term. It can be concluded that adjuvant arthritis deteriorates the adrenergic innervation of the uterus. The effects of exogenous sympathomimetics are shifted, favoring a state of higher contractility. If similar mechanisms are operative in humans, the present results could imply that ß-adrenoceptor agonists are not ideal tocolytics when pregnancy is complicated by generalized inflammation.

Cytotoxic activities of Stachys species.

The cytotoxic activity of Stachys plants and of aucubin and harpagide against MCF7-breast adenocarcinoma, HeLa-cervix adenocarcinoma, A431-skin carcinoma of epithelial origin is reported in this study. Cisplatin and doxorubicin were use as reference compound.

Effects of streptozotocin-induced diabetes on the uterine adrenergic nerve function in pregnant rats. A superfusion study.

Pregnancy-induced diabetes mellitus poses one of the greatest challenges in obstetrical practice. The direct action of diabetes on the myometrial adrenergic functions has not been completely characterized. Accordingly, the present study relates to the impact of experimentally induced diabetes on the presynaptic functions of the rat uterus in relation to gestational age. Experiments were carried out on non-pregnant, early-pregnant (day 7), middle-pregnant (day 14) and late-pregnant (day 21) animals. Diabetes was induced with streptozotocin (60 mg/kg, i.v.) in virgin female or early-pregnant animals (on day 2 for the day 7 experiments and on day 5 for the experiments on the middle and late-pregnant animals). Myometrial samples were utilized for superfusion experiments. After saturation, [3H]noradrenaline perfusate fractions were collected and electric field stimulation was applied to determine the amount of transmitter liberated. Additionally, the total uptake capacity of each sample was assayed. Experimental diabetes decreases the transmitter uptake capacity both in virgin rats and at all stages of pregnancy. In early pregnancy (on day 7), this limitation in uptake is obvious as early as 5 days after the induction of diabetes. In non-pregnant animals, the electrically stimulated transmitter release is inhibited substantially, a similar decrease being observed only at mid-pregnancy (day 14). The present superfusion study proves that experimental diabetes depresses the presynaptic adrenergic functions (both the transmitter uptake and the stimulated release) in the myometrium of the rat. Since the effect of diabetes on the uptake capacity can be detected earlier than for generally accepted markers of peripheral neuropathies, superfusion can be suggested as a sensitive and reliable approach for investigations of hyperglycaemia-related functional deteriorations. We speculate that diabetes-induced functional deterioration of the adrenergic nerves could partially explain the anomalies of the reproductive functions found in diabetic patients if a similar mechanism is operative in humans.

WITHDRAWN: Effects of diabetes on the uterine adrenergic nerve function in pregnant rats. A superfusion study.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Neurochemistry International, doi:10.1016/j.neunet.2005.10.001. The duplicate article has therefore been withdrawn.

Comparison of antioxidant activity in enzyme-independent system of six Stachys species.

The antioxidant activity of six Stachys species native to Hungary in an enzyme-independent lipid-peroxidation system was examined. The methanolic extracts were found to be more effective than the controls alpha-tocopherol succinate and ascorbic acid. The following components were determined by spectrophotometric analysis: hydroxycinnamic acid derivatives, phenols content and flavonoids.

A rat model for functional characterization of pregnancy-induced denervation and postpartum reinnervation in the myometrium and cervix: a superfusion study.

The pregnancy-induced rapid degeneration of the adrenergic nerves innervating the uterus is a well-known but poorly understood phenomenon. Since most of the published investigations were carried out by histological assay, our aim was to describe the loss of the adrenergic function during pregnancy and the re-innervational procedure in the postpartum period. Myometrial and cervical samples from rats were loaded with [3H]noradrenaline and then transferred into a chamber for superfusion. After a wash-out period, fractions were collected. The fifth and fifteenth fraction tissues were stimulated with an electric field. The [3H]noradrenaline contents of the fractions were determined, together with the amount remaining in the tissue. The myometrial [3H]noradrenaline release was substantially decreased in early pregnancy, and absent in the late stage. Differences in release profile were detected between the implantation sites and the interimplantation areas. As a refinement of the results of previous histochemical studies, the noradrenergic functions of the cervix were found to be deeply affected in the early postpartum period. The pregnancy-induced denervational procedure can be followed by means of a superfusional technique after [3H]noradrenaline loading. As our technique is considered to be similar in sensitivity to histological methods, superfusion can be regarded as a model for functional investigations of pregnancy-induced denervation.

Noncompetitive nature of oxytocin antagonists with general structure Mpa(1)Xxx(2)Sar(7)Arg(8).

Eight oxytocin (OT) antagonists with general structure Mpa(1)Sar(7)Arg(8), substituted at position 2 with conformationally constrained and bulky amino acids, were synthesized and pharmacologically tested. Binding affinities and selectivities of compounds for OT, and vasopressin receptor subtypes were investigated. In vitro effects of antagonists were evaluated via inhibition of OT-induced contractions of isolated guinea-pig uterus. The abilities of OT antagonists to inhibit spontaneous contractility in 24 h postpartum rat uterus were investigated. These peptides exhibited pseudoirreversible pharmacological properties, and comprise a novel group of OT antagonists for potential clinical use. Their noncompetitive pharmacological nature can be of therapeutic benefit through a sustained effect on myometrium.

In vitro study of the photocytotoxicity of some hypericin analogs on different cell lines.

In the present study, hypericin analogs with an increased hydrophilic character were synthesized. As chemical modifications alter the lipophilicity/hydrophilicity balance together with the photophysical/chemical background of the molecule the influence of these structural changes on the cellular uptake, retention and subcellular localization in HeLa cells was investigated. Besides, their photocytotoxic effects using three cell lines (HeLa, MCF-7, A431), as well as their plasma protein binding were also assessed. To assess the relative hydrophilic/lipophilic character of hypericin and analogs their retention times were determined on a reversed phase high performance liquid chromatography (C-18) column. The retention time of all the hypericin analogs was < 46 min, except for dibenzyltetramethylhypericin (118 min), while the retention time of hypericin was > 200 min (solvent system: methanol/citrate buffer 30 mM pH 7; 70/30). Hypericin, hexa-, penta- and dibenzyltetramethylhypericin displayed a potent antiproliferative effect at the nanomolar range after photosensitization (3.6 J/cm2). On the contrary, photoactivated tetrasulfonhypericin and fringelite D had no antiproliferative effect on the three cell lines, whereas hypericin polyethylene glycol showed only an intermediate cytotoxic effect on A431 cells. In dark conditions no antiproliferative effect was observed for any photosensitizer. The antiproliferative photo-effect correlated well with the intracellular accumulation as measured using HeLa cells. In general, the photocytotoxic hypericin analogs concentrated to a large extent, while the noncytotoxic compounds were not taken up by the HeLa cells. Furthermore, confocal laser microscopy revealed that all photosensitizers mainly concentrated in the perinuclear region, probably corresponding with Golgi apparatus and the endoplasmic reticulum, except for tetrasulfonhypericin which located at the plasma membrane. In addition, the plasma protein binding studies illustrated that hypericin bind extensively to the low-density lipoproteins, while the other hypericin analogs were mainly bound to heavy proteins (mostly albumin) and to a small extent to low-density lipoproteins.

Antioxidant activity of leaves of Salvia species in enzyme-dependent and enzyme-independent systems of lipid peroxidation and their phenolic constituents.

The protective effects of eleven Salvia species native to Europe against enzyme-dependent and enzyme-independent lipid peroxidation were evaluated. The 50% aqueous methanolic extracts of the leaves of all tested plants were found to be more effective than the positive control alpha-tocopherol acid succinate. The extracts of S. candelabrum, S. ringens, S. tomentosa, S. nemorosa, and S. glutinosa displayed considerable concentration-dependent antioxidative effects that were comparable to those of the medicinal and aromatic plant S. officinalis. The concentrations of flavonoids, hydroxycinnamic acids and total phenolic compounds in each extract were quantified with the aim of clarifying the connection between activity and chemical composition.

In vivo photodynamic activity of hypericin in transitional cell carcinoma bladder tumors.

In a recent clinical study, we showed that hypericin accumulates selectively in urothelial lesions of the bladder following intravesical administration of the compound in patients. This observation infers that hypericin, a potent photosensitizer, could be used as a selective photodynamic therapy (PDT) tool against superficial bladder cancer. In the present study we investigated the in vivo PDT activity of hypericin in transition cell carcinoma (TCC) tumors of the bladder. Both the distribution and tumor PDT response were carried out using subcutaneous heterotopic AY-27 TCC tumors in syngeneic rats. For both PDT and distribution studies, hypericin (1 or 5 mg/kg) was injected intravenously 0.5, 6 or 24 h before PDT or distribution evaluation. The data show that hypericin is a potent photosensitizer in the treatment of TCC tumors in vivo and that the interval between drug administration and photo-irradiation has a dramatic effect on the PDT outcome. Using a 0.5 h interval between drug administration and photo-irradiation the tumor regrowth study indicated that no tumor mass could me measured 9-10 days after PDT. On the contrary, lengthening the time interval between drug administration and photo-irradiation resulted in a gradual loss of PDT efficiency in these tumors. For instance, while the 6 h drug interval protocol produced a moderate PDT activity in which the tumor sizes decreased to about 50% of their original sizes 11-16 days after photo-irradiation, the 24 h interval protocol was even less effective. The distribution data indicate that the PDT efficiency of hypericin in TCC tumors corresponded to the plasma concentrations rather than to the over all concentrations in the tumor. It is therefore conceivable that the mechanism of PDT efficacy of hypericin in TCC tumors is through indirect (vascular effects) rather than through direct effects (cellular destruction) of hypericin in these tumors. In conclusion, our data indicate that hypericin is a potent photosensitizer against AY-27 TCC tumors and that the PDT efficacy of hypericin is largely determined by photosensitizer distribution in the tumor at the time of photo-irradiation.

Photodynamic therapy with hypericin in a mouse P388 tumor model: vascular effects determine the efficacy.

Hypericin, a polycyclic quinone obtained from plants of the Hypericum genus, exhibits strong photodynamic antitumor effects. In the present study, PDT efficacy of hypericin under different conditions was compared in a P388 mouse tumor model. Plasma and tumor drug measurements and assessment of vascular damage by fluorescein dye exclusion were performed to determine the relative contributions of vascular effects and direct tumor cytotoxicity. Furthermore, the influence of modifying tumor oxygenation on PDT effect was also evaluated. Study of PDT efficacy and tissue distribution revealed that PDT efficacy was more dependent on plasma concentration than tumor drug level. Fluorescein dye exclusion indicated the complete microvascular occlusion in the tumor and surrounding skin immediately after effective PDT treatments, while only a limited vascular occulation was observed after non-effective PDT treatment. It was found that neither tumor hypoxia induced by hydralazine nor increasing tumor oxygenation achieved by nicotinamide could significantly affect the effectiveness of various PDT protocols. These results suggest that tumor vasculature damage might be the primary mechanism of hypericin-mediated PDT effect. The existence of this potent secondary vascular effect is likely to account for the inability of tumor oxygenation modifiers to affect tumor response after PDT with hypericin.

Protective effects of the aerial parts of Salvia officinalis, Melissa Officinalis and Lavandula angustifolia and their constituents against enzyme-dependent and enzyme-independent lipid peroxidation.

The antioxidant effects of aqueous methanolic extracts from three medicinal Lamiaceae species were investigated in enzyme-dependent and enzyme-independent lipid peroxidation systems. All these extracts caused a considerable concentration-dependent inhibition of lipid peroxidation. Phenolic components present in the plant extracts were evaluated for antioxidant activity and were found effective in both tests. Their concentrations in each extract were determined by TLC-densitometry.

In vivo effects of 2-substituted [Mpa1Sar7Arg8]-oxytocin antagonists on postpartum rat.

A set of oxytocin antagonists consisting of [Mpa1Sar7Arg8]-oxytocin substituted by various conformationally restricted or bulky D amino acids at position 2 were synthetized and biologically tested. In in vivo pharmacological investigations, the effects of these peptides were examined on the spontaneous motor activity of postpartum rat. Three of the newly prepared peptides proved at least as effective in inhibiting uterine contractions as clinically investigated atosiban.

Correlation between alpha1/beta-adrenoceptor ratio and spontaneous uterine motor activity in the post-partum rat.

The spontaneous uterine motor activity of the post-partum rat was investigated in parallel with the in-vitro determination of the density of the alpha1 and beta-adrenergic receptors of the myometrium. The in-vivo experiments were performed by an improved method, using a Millar catheter fitted with a latex microballoon. The spontaneous contractility of the post-partum rat uterus was found to be highest 24 h after delivery, indicating that this time is the most suitable for pharmacological examinations of tocolytic agents. A very close correlation was found between the results of the in-vivo experiments and the alpha1/beta-adrenergic receptor ratio assessed by an in-vitro receptor assay, thus indicating that the state of the adrenergic receptor system fundamentally determines the contractility of the uterus. This conclusion is supported by the fact that the pharmacological sensitivity of the rat uterus to prazosin and fenoterol changed as a function of the post-partum time in accordance with the alpha1/beta-adrenoceptor ratio. These results and the relevant data available reveal a crucially important role of an alpha1-adrenoceptor-mediated process, implicating alpha1-blockers as theoretically potent agents for inhibition of premature uterine contractions.

Evidence of non-synaptic regulation of postpartum uterine contractility in the rat.

Myometrial tissue rings from postpartum rats (24 h after delivery) were studied in vitro by electric field stimulation, and the alpha1/beta2-adrenoceptor ratio was determined by a radioligand binding technique. Pregnancy-denervated uterine rings were stimulated by long-duration pulses (100 ms). The contractions were inhibited by beta2-agonists (terbutaline and fenoterol) and alpha-antagonists (phentolamine, urapidil and yohimbine) in a concentration-dependent manner. Their effects were not altered by the adrenergic neuron-blocking agent bretylium. The alpha-antagonists (except phentolamine) elicited the same maximal inhibition as the beta2-agonists. Receptor assays revealed that the alpha1/beta2 ratio was about 2 in the measured uteri. It was concluded that the inhibitory effects of alpha-antagonists and beta2-agonists are mediated via non-synaptic adrenoceptors of the denervated postpartum rat uterus. The same inhibitory activity could be explained by the greater amount of alpha-receptors. It is believed that this is the first functional proof of the existence of non-synaptic alpha1-adrenoceptors in smooth muscle.

Are alpha-adrenergic antagonists potent tocolytics? In vivo experiments on postpartum rats.

The aim of this study was to investigate the effects of alpha-adrenergic antagonists on the motor activity of the postpartum uterus of the rat in vivo. Intrauterine pressure was assessed by means of a Millar catheter fitted with a latex microballoon. Some of the tested compounds (urapidil, yohimbine, phentolamine, benoxathian and prazosin) decreased the uterine activity to a significant extent (57.4-67.4%). However, none of the investigated alpha receptor blockers exerted the same effect as beta-adrenergic agonists. Our results suggest that alpha-adrenergic antagonists could possibly be used as an alternative to beta-adrenergic agonists in clinical tocolysis after an appropriate clinical evaluation.

[Effects of calcium channel blockers on electrical and mechanical responses of rat phrenic nerve-diaphragm preparations]. / Kalcium-csatorna blokkolók hatása patkány nervus phrenicus-diaphragma preparátum elektromos és mechanikus válaszára.

The effects of diltiazem, prenylamine and verapamil on the electrical and mechanical responses of a rat phrenic nerve-diaphragm preparation were investigated as a function of the indirect stimulating frequency. The amplitude of the muscle compound action potential was depressed by all three drugs with stimulation between 3 and 50 Hz. The contractile force of the diaphragm was increased by these drugs in concentrations of 3 x 10(-8)-3 x 10(-5) mol/l at low frequency (3-10 Hz), but the contractility was decreased at higher concentrations (above 3 x 10(-5) mol/l). In contrast, a facilitating effect was not observed at a higher stimulating frequency (above 20 Hz), but a concentration depressant effect did develop above 1 x 10(-6) mol/l. The literature data and our results suggest that low concentrations of calcium channel blocking agents can not influence the trigger calcium current at low frequency (under 15 Hz), but calcium liberation and the contractile force are increased by these drugs. The intracellular calcium liberation triggered by the calcium current or the muscle force was inhibited by these drugs at higher concentrations. Above 15 Hz, the liberated intracellular calcium is insufficient for the contraction, so an adequate quantity of calcium is obtained from the extracellular space. It is suggested that an adequate calcium uptake is inhibited at low concentrations of these drugs at higher frequency, and the contractility is thereby inhibited.