Concentration-dependent Way Research Articles

Interaction of Green Tea Catechins with Organic Anion Transporting Polypeptides

Organic anion transporting polypeptides (OATPs) are multispecific transporters that mediate the uptake of numerous drugs and xenobiotics into cells. Here, we examined the effect of green tea (Camellia sinensis) flavanols on the function of three OATPs expressed on the basolateral membrane of human hepatocytes. Uptake of the model substrate estrone‐3‐sulfate was measured using CHO cells stably expressing OATP1B1, OATP1B3, or OATP2B1 in the absence and presence of the four most abundant flavanols found in green tea. Uptake by OATP1B1 and OATP2B1 was inhibited by epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) in a concentration dependent way. In contrast, OATP1B3‐mediated uptake of estrone‐3‐sulfate was strongly stimulated by EGCG (5‐fold increase, EC50 = 10.9 μM). The effect of EGCG on OATP1B3 was also studied with additional substrates: uptake of estradiol‐17β‐glucuronide was not affected, while uptake of Fluo‐3 was inhibited (IC50 of 8.3μM). These results indicate that two of the major flavanols found in green tea have a substantial effect upon the function of OATPs expressed in hepatocytes and can potentially alter the pharmacokinetics of drugs and other OATP substrates. These substrate dependent effects of EGCG on OATP1B3 seem to affect different transport/binding sites. In the future, we will use LC‐MS/MS analysis to identify whether these compounds are directly transported by OATPs.

New NO donor inhibits contraction of smooth muscle cell through activation of guanylyl‐cyclase and sarcoplasmic reticulum Ca+2‐ATPase (SERCA).

This study aimed to investigate the pathways involved in the vasodilatation induced by the new NO donor cis‐[Ru(bpy)2(py)(NO2)](PF6)3 (PY) synthesized in our laboratory. Vascular reactivity to PY was studied in denuded rat aorta pre‐contracted with phenylephrine (Phe) or 60mM KCl. Concentration‐effect curves for PY were constructed in the presence or in the absence of the selective inhibitor of soluble guanylyl‐ cyclase (sGC) (ODQ), superoxide scavenger (Tiron) and SERCA inhibitor (Thapsigargin, TG). It was also constructed concentration‐effect curves for CaCl2 under stimulation with 100nM Phe with or without PY. NO was released by the compound only in presence of the aortic tissue as detected by amperometry with a selective sensor. The NO donor reached the maximum aortic rings relaxation (ME) in 210 seconds. PY induced complete relaxation of the aortic rings pre‐contracted with Phe or KCl in a concentration‐dependent way but the ME and potency (pD2) values were lower in KCl than in Phe‐contracted aorta. These responses were inhibited by ODQ and TG. The contractions to increased concentrations of extracellular Ca2+ were abolished by the 3 uM PY. The effect of PY was blocked by ODQ or TG. Our results show that the NO released from PY in the tissue induces relaxation due to the activation of the enzymes sGC and SERCA. It also inhibits Ca2+ influx and consequent contractile response. Financial Support: Fapesp and CNPq.

Cytotoxic and apoptotic effects of single and mixed oxides of β-sitosterol on HepG2-cells

While health implications caused by cholesterol oxidation products (COPs) seem to be generally accepted, research on phytosterol oxidation products (POPs) is still limited. Since POPs are commercially not available knowledge on their toxic activities is mainly derived from blends instead of pure compounds. Therefore the aim of the present study was to examine the cytotoxicity of three individual oxidation products of β-sitosterol, 7-ketositosterol, 7β-OH-sitosterol, 7α-OH-sitosterol, a mixture of 6β-OH-3-keto-sitosterol/6α-OH-3-keto-sitosterol (ratio 4:3) and a mixture of polar oxides towards HepG2-cells. All tested compounds were found to reduce cell viability in a significant and concentration dependent way, particularly 7-keto- and 7α-OH-sitosterol showed to be highly active. Only for 7-ketositosterol an increase in early apoptotic cells was observed. Enhancement of O 2 - production was assessed for all oxides, whereas malondialdehyd (MDA) levels were increased by 7-keto- and 7α-OH-sitosterol only. However, cell death did not appear to be necessarily dependent on the generation of oxidative stress. Further no DNA strand breaks were observed with the COMET assay. By assessing the accumulation of single oxidation products in the cells a link between higher proportions of oxides inside the cells and their cytotoxic potential could be found.

Interactions of mast cell degranulating peptides with model membranes: A comparative biophysical study

In the last decade, there has been renewed interest in biologically active peptides in fields like allergy, autoimmune diseases and antibiotic therapy. Mast cell degranulating peptides mimic G-protein receptors, showing different activity levels even among homologous peptides. Another important feature is their ability to interact directly with membrane phospholipids, in a fast and concentration-dependent way. The mechanism of action of peptide HR1 on model membranes was investigated comparatively to other mast cell degranulating peptides (Mastoparan, Eumenitin and Anoplin) to evidence the features that modulate their selectivity. Using vesicle leakage, single-channel recordings and zeta-potential measurements, we demonstrated that HR1 preferentially binds to anionic bilayers, accumulates, folds, and at very low concentrations, is able to insert and create membrane spanning ion-selective pores. We discuss the ion selectivity character of the pores based on the neutralization or screening of the peptides charges by the bilayer head group charges or dipoles.

Exploring the binding site of acetogenin in the ND1 subunit of bovine mitochondrial complex I

125I-labeled ( trifluoromethyl)phenyl diazirinyl acetogenin, [ 125I]TDA, a photoaffinity labeling probe of acetogenin, photo-cross-links to the ND1 subunit of bovine heart mitochondrial NADH–ubiquinone oxidoreductase (complex I) with high specificity [ M. Murai, A. Ishihara, T. Nishioka, T. Yagi, and H. Miyoshi, (2007) The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I, Biochemistry 46 6409–6416.]. To identify the binding site of [ 125I]TDA in the ND1 subunit, we carried out limited proteolysis of the subunit cross-linked by [ 125I]TDA using various proteases and carefully analyzed the fragmentation patterns. Our results revealed that the cross-linked residue is located within the region of the 4th to 5th transmembrane helices (Val144–Glu192) of the subunit. It is worth noting that an excess amount of short-chain ubiquinones such as ubiquinone-2 (Q 2) and 2-azido-Q 2 suppressed the cross-linking by [ 125I]TDA in a concentration-dependent way. Although the question of whether the binding sites for ubiquinone and different inhibitors in complex I are identical remains to be answered, the present study provided, for the first time, direct evidence that an inhibitor (acetogenin) and ubiquinone competitively bind to the enzyme. Considering the present results along with earlier photoaffinity labeling studies, we propose that not all inhibitors acting at the terminal electron transfer step of complex I necessarily bind to the ubiquinone binding site itself.

Inhibitory Effect of Ghrelin on Nicotine-induced VCAM-1 Expression in Human Umbilical Vein Endothelial Cells

Endothelial dysfunction is thought to be a major cause of vascular injury in smokers. Ghrelin is a recently discovered peptide that plays a modulatory role in atherosclerosis. However, it is unknown how ghrelin regulates nicotine-induced vascular cell adhesion molecule-1 (VCAM-1) expression. We examined nicotine-induced VCAM-1 expression in human umbilical vein endothelial cells pretreated with ghrelin and detected the activity of protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), and nuclear factor (NF)-kappaB. Our study showed that ghrelin inhibited nicotine-induced VCAM-1 expression in human umbilical vein endothelial cells in a concentration-dependent and time-dependent way. We also found that ghrelin inhibited nicotine-induced PKC, p38 MAPK, and NF-kappaB activation. The results suggest that ghrelin inhibits nicotine-induced VCAM-1 expression, and PKC, p38 MAPK, and NF-kappaB play active roles in that process. Exogenous ghrelin may provide a possible approach for preventing or reversing atherosclerosis in smokers.

Metabolic profiling and biological capacity of Pieris brassicae fed with kale (Brassica oleracea L. var. acephala)

Phenolic and organic acid profiles of aqueous extracts from Pieris brassicae material and the host kale (Brassica oleracea L. var. acephala) leaves were determined by HPLC/UV–DAD/MSn-ESI and HPLC–UV, respectively. The identified phenolics included acylated and nonacylated flavonoid glycosides, hydroxycinnamic acyl gentiobiosides, and sulphate phenolics. Kale exhibited the highest content (11g/kg lyophilized extract), while no phenolics were identified in the butterflies or exuviae. Nine different organic acids were characterized in the materials, with kale showing the highest amount (112g/kg lyophilized extract). With the exception of the exuviae extract, the rest were screened for bioactivity. Using spectrophotometric microassays, all exhibited antiradical capacity against DPPH and NO in a concentration-dependent way, whereas only kale and excrement extracts were active against superoxide. All displayed activity on intestinal smooth muscle, albeit with distinct relaxation–contraction profiles. Larvae and butterfly extracts were more efficacious for intestinal relaxation than was kale extract, whereas excrement extract evoked only contractions, thus evidencing their different compositions. Collectively, these results show that P. brassicae sequesters and metabolizes kale’s phenolic compounds. Moreover, the extract’s bioactivities suggest that they may constitute an interesting source of bioactive compounds whose complex chemical structures preclude either synthesis or isolation.

Coriaria Lactone Increased the Intracellular Level of Calcium through the Voltage-gated Calcium Channels in Rat Hippocampal Neurons

To investigate the effects of Coriaria Lactone (CL), an epileptogenic substance, on intracellular levels of calcium ([Ca(2+)](i)) and physiological properties of voltage-gated calcium channels (VGCCs). Ratiometric calcium imaging using Fura Red and whole-cell voltage patch-clamp technique were explored on freshly isolated rat hippocampal neurons exposed to CL. Coriaria Lactone increased [Ca(2+)](i) from 118 +/- 21 to 440 +/- 35 nM; VGCCs and calcium influx through NMDA receptor served as the main routes of entry. Coriaria Lactone could enhance both Low voltage activated (LVA) and High voltage activated calcium currents in a concentration-dependent way, and its effect on LVA current was more potent (about 60%). The increased calcium currents were accompanied by the shift of voltage-dependent steady-state inactivation to more positive potentials. These effects of CL, especially its impact on LVA current, could activate different calcium-dependent signaling pathways, and influence cellular excitable properties as well, which might play an important role in CL's epileptogenic process.

Effects of Acetylsalicylic Acid on Increase of Fibrin Network Porosity and the Consequent Upregulation of Fibrinolysis

Our earlier study in vivo showed that a lower dose of acetylsalicylic acid (ASA) brought greater enhancement in fibrin gel permeability (Ks) than a higher dose. To assess whether this finding related to modifications of fibrinogen clotting property by ASA, purified fibrinogen was incubated with ASA and/or salicylic acid (SA). The fibrinogen product was examined. Fibrinogen "clotting time" was not affected. Shortening of fibrin clot "lysis time" paralleled the increase of fibrin network porosity demonstrated by measurements of liquid permeability (Ks), fibrin fiber thickness, and 3-dimensional microscopic image, in a low ASA concentration-dependent way. Ks levels were not altered by SA alone but significantly decreased in samples treated by both where the concentrations were low for ASA and high for SA. In conclusion, ASA at the concentrations used did not influence the rate of fibrinogen gelation by thrombin. However, assembly of fibrin monomers was most probably altered, leading to enhancement of fibrin fiber thickness. A looser network was constructed by the thicker fibrin fibers, which benefits fibrinolysis. According to the known mechanism that SA interferes with ASA in preventing acetylation of platelet's proteins, an explanation for the low ASA concentration-dependent effects on fibrin network structure may be that fewer molecules of SA-the hydrolytic product of ASA-are generated from lower doses of ASA, which block acetylation of fibrinogen to a smaller extent and thus more significantly impair fibrin formation.

A Novel Surface Modification Using Tissue Factor Reconstituted in Phospholipid Vesicles for the Activation of Blood Coagulation

We describe a novel method to immobilize recombinant human tissue factor (rhTF) reconstituted in phospholipid vesicles. The rhTF vesicles were immobilized in a multilayer vesicle structure using cholesterol–DNA tethers spontaneously inserted into the lipid membrane. The properties of the rhTF vesicle surface modification were characterized by surface plasmon resonance biosensor technology. As an application of this surface modification, we investigated its use as a blood coagulation activating surface. The coagulation activating capacity of the surface modification was tested by exposure to human whole blood in a flow chamber. No increase in rhTF levels in the blood was found after passage through the flow chamber, indicating that the rhTF surface modification was stable. Thrombin–antithrombin (TAT) and prothrombin fragment (PF) 1 + 2 levels increased after exposure to the surface, and decreased in a concentration-dependent way upon addition of melagatran (a direct thrombin inhibitor), i.e., coagulation activity triggered by rhTF could be suppressed by anticoagulation. The results with this new thrombogenic surface are promising, and will be further developed into a useful tool for coagulation related investigations, e.g., characterization of anticoagulants and biomaterials.

Furanoditerpenes from Pterodon pubescens benth with selective in vitro anticancer activity for prostate cell line

Activity guided fractionation of Pterodon pubescens Benth. methylene chloride-soluble fraction afforded novel 6α-acetoxi 7β-hydroxy-vouacapan 1 and four known diterpene furans 2, 3, 4, 5. The compounds were evaluated for in vitro cytotoxic activities against human normal cells and tumour cell lines UACC-62 (melanoma), MCF-7 (breast), NCI-H460 (lung, non-small cells), OVCAR-03 (ovarian), PC-3 (prostate), HT-29 (colon), 786-0 (renal), K562 (leukemia) and NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance). Results were expressed by three concentration dependent parameters GI50 (concentration that produces 50% growth inhibition), TGI (concentration that produces total growth inhibition or cytostatic effect) and LC50 (concentration that produces -50% growth, a cytotoxicity parameter). Also, in vitro cytotoxicity was evaluated against 3T3 cell line (mouse embryonic fibroblasts). Antiproliferative properties of compounds 1, 4 and 5 are herein reported for the first time. These compounds showed selectivity in a concentration-dependent way against human PC-3. Compound 1 demonstrated selectivity 26 fold more potent than the positive control, doxorubicin, for PC-3 (prostrate) cell line based on GI50 values, causing cytostatic effect (TGI value) at a concentration fifteen times less than positive control. Moreover comparison of 50% lethal concentration (LC50 value) with positive control (doxorubicin) suggested that compound 1 was less toxic.

SCAVENGING ACTIVITY OF DICLOFENAC: INTERACTION WITH ABTS RADICAL CATION AND PEROXYL RADICALS

ABSTRACT The scavenging activity of diclofenac was estimated by its capability to bleach ABTS radical cation, and ORAC (oxygen radical absorbance capacity) methodology. In addition, GC-MS technique was used to establish if the lactamic derivative of diclofenac was formed as final product in the interaction with peroxyl radicals. Diclofenac bleached ABTS radical cation in a concentration-dependent way. In ORAC assays, diclofenac inhibited the consumption of fluorescein and c-phycocyanin mediated by peroxyl radicals. An ORAC-fluorescein, and ORAC-c-phycocyanin value of 0.3, and 0.004 was estimated, respectively. From the downcurvature observed in Stern-Volmer-like plots, we postulated that the low protection of c-phycocyanin is related to secondary damage of c-phycocyanin by the secondary radical of diclofenac. By GC-MS studies only a 2,6-dichloroaniline derivative was detected as product after the reaction with peroxyl radicals. Key Words: Diclofenac, fluorescein, c-phycocyanin, ORAC, scavenging activity.

Platelet-derived growth factor BB promotes the migration of bone marrow-derived mesenchymal stem cells towards C6 glioma and up-regulates the expression of intracellular adhesion molecule-1

Recent studies have indicated that bone marrow-derived mesenchymal stem cells (BMSCs) have the capacity of migrating towards gliomas. However, few data are available about the molecular mechanism responsible for this migratory capacity. The aim of our study was to investigate the role of platelet-derived growth factor BB (PDGFBB) in the migration of BMSCs towards C6 glioma and evaluate the effect of PDGFBB on the migrating capacity and intercellular adhesion molecule-1 (ICAM-1) expression of BMSCs. The chemokinetic activity of BMSCs in response to C6 glioma-conditioned medium and recombinant rat PDGFBB was analyzed by in vitro migration assay. The effect of PDGFBB on the expression of ICAM-1 was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. Our data showed that C6 glioma-conditioned medium significantly increased the migration of BMSCs, which could be partially blocked by a PDGFBB neutralizing antibody. Recombinant rat PDGFBB enhanced the migration of BMSCs in a concentration-dependent way from 5 to 50 ng/ml. Moreover, RT-PCR and immunofluorescence showed that 12 h of 20 ng/ml PDGFBB incubation could up-regulate the ICAM-1 expression of BMSCs. Our data also revealed that SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), significantly decreased the PDGFBB-induced migration and ICAM-1 expression of BMSCs. These results demonstrate that PDGFBB contributes to the migration of BMSCs towards C6 glioma and up-regulates the expression of ICAM-1, and that p38MAPK is an important signaling molecule correlating with the signal transduction of PDGFBB-induced migration and ICAM-1 expression of BMSCs.

ROS scavenging capacity and neuroprotective effect of α-mangostin against 3-nitropropionic acid in cerebellar granule neurons

α-Mangostin is a xanthone with antioxidant properties isolated from mangosteen fruit. The reactive oxygen species (ROS) scavenging capacity and the potential protective effect of α-mangostin against the mitochondrial toxin 3-nitropropionic acid (3-NP) in primary cultures of cerebellar granule neurons (CGNs) were studied in the present work. It was found that α-mangostin was able to scavenge in a concentration-dependent way singlet oxygen, superoxide anion and peroxynitrite anion. In contrast, α-mangostin was unable to scavenge hydroxyl radicals and hydrogen peroxide. Furthermore, α-mangostin was able to ameliorate in a concentration-dependent way the neuronal death induced by 3-NP. This protective effect was associated with an amelioration of 3-NP-induced reactive oxygen species formation. It is concluded that α-mangostin is able to scavenge directly several ROS and has a neuroprotective effect against 3-NP in primary cultures of CGNs, which is associated with its ability to ameliorate 3-NP-induced ROS production.

Material basis for inhibition of Dragon’s Blood on evoked discharges of wide dynamic range neurons in spinal dorsal horn of rats

In vivo experiments were designed to verify the analgesic effect of Dragon's Blood and the material basis for this effect. Extracellular microelectrode recordings were used to observe the effects of Dragon's Blood and various combinations of the three components (cochinchinenin A, cochinchinenin B, and loureirin B) extracted from Dragon's Blood on the discharge activities of wide dynamic range (WDR) neurons in spinal dorsal horn (SDH) of intact male Wistar rats evoked by electric stimulation at sciatic nerve. When the Hill's coefficients describing the dose-response relations of drugs were different, based on the concept of dose equivalence, the equations of additivity surfaces which can be applied to assess the interaction between three drugs were derived. Adopting the equations and Tallarida's isobole equations used to assess the interaction between two drugs with dissimilar dose-response relations, the effects produced by various combinations of the three components in modulating the evoked discharge activities of WDR neurons were evaluated. Results showed that Dragon's Blood and its three components could inhibit the evoked discharge frequencies of WDR neurons in a concentration-dependent way. The Hill's coefficients describing dose-response relations of three components were different. Only the combined effect of cochinchinenin A, cochinchinenin B and loureirin B was similar to that of Dragons Blood. Furthermore, the combined effect was synergistic. This investigation demonstrated that through the synergistic interaction of the three components Dragon's Blood could interfere with the transmission and processing of pain signals in spinal dorsal horn. All these further proved that the combination of cochinchinenin A, cochinchinenin B, and loureirin B was the material basis for the analgesic effect of Dragon's Blood.

Salt Processing in Larval Drosophila: Choice, Feeding, and Learning Shift from Appetitive to Aversive in a Concentration-Dependent Way

Sodium and chloride need to be ingested and cannot be stored. Therefore, choice of habitat and diet as related to NaCl needs to be tightly regulated. We thus expect that the behavioral effects of salt are organized according to its concentration. Here, we comparatively “fingerprint” the reflex releasing (in choice and feeding experiments) versus the reinforcing effects of sodium chloride (“salt”) in terms of their concentration dependencies, using larval Drosophila. Qualitatively, we find that the behavioral effects of salt in all 3 assays are similar: choice, feeding, and reinforcing effect all change from appetitive to aversive as concentration is increased. Quantitatively, however, the appetitive effects for choice and feeding share their optimum at around 0.02 M, whereas the dose–response curve for the reinforcing effect is shifted by more than one order of magnitude toward higher concentrations. Interestingly, a similar shift between these 2 kinds of behavioral effect is also found for sugars (Schipanski et al. 2008). Thus, for salt and for sugar, the sensory-to-motor system is more sensitive regarding immediate, reflexive behavior than regarding reinforcement. We speculate that this may partially be due to a dissociation of the sensory pathways signaling toward either reflexive behavior or internal reinforcement.

Human dendritic cell activities are modulated by the omega-3 fatty acid, docosahexaenoic acid, mainly through PPARγ:RXR heterodimers: comparison with other polyunsaturated fatty acids

There is accumulating evidence that omega-3 fatty acids may modulate immune responses. When monocytes were differentiated to dendritic cells (DCs) in the presence of docosahexaenoic acid (DHA), the expression of costimulatory and antigen presentation markers was altered in a concentration-dependent way, positively or negatively, depending on the markers tested and the maturation stage of the DCs. Changes induced by eicosapentaenoic acid and linoleic acid were similar but less intense than those of DHA, whereas oleic acid had almost no effect. DHA-treated, mature DCs showed inhibition of IL-6 expression and IL-10 and IL-12 secretion, and their lymphoproliferative stimulation capacity was impaired. The phenotypic alterations of DCs induced by DHA were similar to those already reported for Rosiglitazone (Rosi), a peroxisome proliferator-activated receptor gamma (PPAR gamma) activator, and the retinoid 9-cis-retinoic acid (9cRA), a retinoid X receptor (RXR) activator. Moreover, DHA induced the expression of PPAR gamma target genes pyruvate dehydrogenase kinase-4 and aP-2 in immature DCs. The combination of DHA with Rosi or 9cRA produced additive effects. Furthermore, when DCs were cultured in the presence of a specific PPAR gamma inhibitor, all of the changes induced by DHA were blocked. Together, these results strongly suggest that the PPAR gamma:RXR heterodimer is the pathway component activated by DHA to induce its immunomodulatory effect on DCs.

Salvinorin A modulates the neurotransmitters release from different mouse brain areas through presynaptic metabotropic receptors activation

Salvinorin-A is a component of Salvia divinorum, displaying psychoactive and hallucinogenic properties in humans. Recent literature suggests that this compound can act as a potent and selective opioid receptor agonist, having high affinity towards the K–opioid receptor subtype. The aim of our study was to investigate whether and to what extent this compound can alter the release of neurotransmitters from isolated nerve terminals (synaptosomes) prepared from mouse hippocampus (HC), striatum (STR) and prefrontal cortex (PFC). The experimental approach used, i.e. the up-down superfusion of a synaptosomal monolayer, is considered a method of choice to investigate presynaptic mechanisms of control of neurotransmitter release. We focused on the release of prelabelled [3H]noradrenaline ([3H]NA) and [3H]serotonin ([3H]5HT) from rat HC synaptosomes and of [3H]dopamine ([3H]DA) from PFC and STR synaptosomes. Salvinorin-A failed to affect the spontaneous release of all the three amines, but significantly modified the release of these neurotransmitters induced by transient (90s) exposure to 12 mM K+. In particular, Salvinorin-A potentiated in a concentration-dependent manner the K+-evoked release of [3H]NA, while it inhibited, always in a concentration-dependent way the release of [3H]5HT and of [3H]DA. Both facilitation and inhibiton of amine exocytosis was prevented by entrapping into synaptosomes the Pertussis toxin, suggesting the primary involvement in these effects of G-protein coupled receptors sensitive to this toxin. These results may be relevant to the comprehension of the molecular mechanisms involved in the central effects induced by this natural product.

Cytotoxic and genotoxic effects of tambjamine D, an alkaloid isolated from the nudibranch Tambja eliora, on Chinese hamster lung fibroblasts

Marine organisms have been shown to be potential sources of bioactive compounds with pharmaceutical applications. Previous chemical investigation of the nudibranch Tambja eliora led to the isolation of the alkaloid tambjamine D. Tambjamines have been isolated from marine sources and belong to the family of 4-methoxypyrrolic-derived natural products, which display promising immunosuppressive and cytotoxic properties. Their ability to intercalate DNA and their pro-oxidant activity may be related to some of the biological effects of the 4-methoxypyrrolic alkaloids. The aim of the present investigation was to determine the cytotoxic, pro-oxidant and genotoxic properties of tambjamine D in V79 Chinese hamster lung fibroblast cells. Tambjamine D displayed a potent cytotoxic effect in V79 cells (IC50 1.2 μg/mL) evaluated by the MTT assay. Based on the MTT result, V79 cells were treated with different concentrations of tambjamine D (0.6, 1.2, 2.4 and 4.8 μg/mL). After 24 h, tambjamine D reduced the number of viable cells in a concentration-dependent way at all concentrations tested, assessed by the trypan blue dye exclusion test. The hemolytic assay showed that the cytotoxic activity of tambjamine D was not related to membrane disruption (EC50 > 100 μg/mL). Tambjamine D increased the number of apoptotic cells in a concentration-dependent manner at all concentrations tested according to acridine orange/ethidium bromide staining, showing that the alkaloid cytotoxic effect was related to the induction of apoptosis. MTT reduction was stimulated by tambjamine D, which may indicate the generation of reactive oxygen species. Accordingly, treatment of cells with tambjamine D increased nitrite/nitrate at all concentrations and TBARS production starting at the concentration corresponding to the IC50. Tambjamine D, also, induced DNA strand breaks and increased the micronucleus cell frequency as evaluated by comet and micronucleus tests, respectively, at all concentrations evaluated, showing a genotoxic risk induced by tambjamine D.

Evaluation of wound healing properties of Arrabidaea chica Verlot extract

Ethnopharmacological relevance Arrabidaea chica Verlot. (Bignoniaceae), popularly known as Crajiru, has been traditionally used as wound healing agent. Aim of the study Investigate in vitro and in vivo healing properties of Arrabidaea chica leaves extract (AC). Materials and methods AC was evaluated in vitro in fibroblast growth stimulation (0.25–250 μg/mL) and collagen production stimulation (250 μg/mL) assays. Allantoin (0.25–250 μg/mL) and vitamin C (25 μg/mL) were used as controls respectively. DPPH and Folin-Ciocalteau assays were used for antioxidant evaluation, using trolox (0.25–250 μg/mL) as reference antioxidant. To study wound healing properties in rats, AC (100 mg/mL, 200 μL/wound/day) was topically administered during 10 days and wound area was evaluated every day. Allantoin (100 mg/mL, 200 μL/wound/day) was used as standard drug. After treatment, wound sites were removed for histopathological analysis and total collagen determination. Results AC stimulated fibroblast growth in a concentration dependent way (EC 50 = 30 μg/mL), increased in vitro collagen production and demonstrated moderate antioxidant capacity. In vivo, AC reduced wound size in 96%, whereas saline group showed only 36% wound healing. Conclusion AC efficiency seems to involve fibroblast growing stimulus and collagen synthesis both in vitro and in vivo, beyond moderate scavenging activity, corroborating Crajiru folk use.