Non-clinical investigations about cytotoxic and anti-platelet activities of gamma-terpinene.

Gamma-terpinene (γ-TPN) is a cyclohexane monoterpene isolated from plant essential oils, such as tea tree (Melaleuca alternifolia), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris Marchand), and eucalyptus (Eucalyptus sp.). Terpenes are widely studied molecules pharmacologically active on the cardiovascular system, hemostasis, and antioxidant actions. Herein, it was investigated the cytotoxic and antiplatelet activity of γ-TPN using different non-clinical laboratory models. For in silico evaluation, the PreADMET, SwissADME, and SwissTargetPrediction softwares were used. Molecular docking was performed using the AutoDockVina and BIOVIA Discovery Studio databases. The cytotoxicity of γ-TPN was analyzed by the MTT assay upon normal murine endothelial SVEC4-10 and fibroblast L-929 cells. Platelet aggregation was evaluated with platelet-rich (PRP) and platelet-poor (PPP) plasma from spontaneously hypertensive rats (SHR), in addition to SVEC4-10 cells pre-incubated with γ-TPN (50, 100, and 200 µM) for 24 h. SHR animals were pre-treated by gavage with γ-TPN for 7 days and divided into four groups (negative control, 25, 50, and 100 mg/kg). Blood samples were collected to measure nitrite using the Griess reagent. Gamma-TPN proved to be quite lipid-soluble (Log P = +4.50), with a qualified profile of similarity to the drug, good bioavailability, and adequate pharmacokinetics. It exhibited affinity mainly for the P2Y12 receptor (6.450 ± 0.232 Kcal/mol), moderate cytotoxicity for L-929 (CC50 = 333.3 µM) and SVEC 4-10 (CC50 = 366.7 µM) cells. The presence of γ-TPN in SVEC 4-10 cells was also able to reduce platelet aggregation by 51.57 and 44.20% at lower concentrations (50 and 100 µM, respectively). Then, γ-TPN has good affinity with purinergic receptors and an effect on the reversal of platelet aggregation and oxidative stress, being promising and safe for therapeutic targets and subsequent studies on the control of thromboembolic diseases.

Alpha-terpineol prevents myocardial damage against isoproterenol-MI induced in Wistar-Kyoto rats: new possible to promote cardiovascular integrity.

Alpha-terpineol (TPN) is one of the major components of the resin obtained from Protium heptaphyllum. This plant has been utilized as medicine by Brazilian indigenous tribes to treat cardiovascular diseases. Scientific reports have shown that the TPN possesses vasorelaxant and antihypertensive effects. This study was conducted to assess the cardioprotective action of TPN against isoproterenol (ISO)-induced cardiotoxicity. Wistar rats were randomly divided into six groups. Rats were orally administered with TPN (25, 50, and 75 mg/kg, respectively) for 15 days, and ISO was administered (85 mg/kg, subcutaneously) on the 14th and 15th days. At the end of the experiment, the hemodynamic, baroreflex test, ECG, biochemical, histological, and morphometric changes were monitored from control and experimental groups, i.e., on the 15th day. ISO-induced myocardial infarcted rats showed an increase in mortality rates, cardiac marker enzymes, tachycardia, hypertrophy, myocardium necrosis, edema, hemorrhagic areas, infiltration of inflammatory cells like lymphocytes, and increased myocardial infarct size. However, pretreatment with TPN significantly inhibited these effects of ISO. The histopathological findings obtained for the myocardium further confirmed the biochemical results. Thus, the present study provides evidence for the efficacy of TPN against ISO-induced myocardial infarction in rats.

Alpha-terpineol complexed with beta-cyclodextrin reduces damages caused by periodontitis in rats.

BACKGROUND AND OBJECTIVE: This study aimed to assess the effectiveness of the treatment with alpha-terpineol (αTPN) complexed with beta-cyclodextrin (ßCD) on oral, blood, and hepatic parameters in ligature-induced periodontitis. MATERIAL AND METHODS: Forty female rats were distributed among the following groups: control (vehicle solution), periodontitis (ligature + vehicle solution), 5 mg/kg of αTPN-ßCD (ligature), and 25 mg/kg of αTPN-ßCD (ligature). Compounds were administered daily via intraperitoneal injection over a 20-day period. Periodontitis was induced with the bilateral insertion of ligatures around the first lower molars of each rat. Oral parameters, as well as blood biomarkers, were measured: histopathological assessment of the hepatic tissue was carried out using light and transmission electron microscopy. RESULTS: The treatment with αTPN-ßCD significantly improved several oral parameters and blood biomarkers in comparison with rats with periodontitis. In addition, the treatment with αTPN-ßCD significantly ameliorated the steatosis score and reduced the number of lipid droplets and the amount of foamy cytoplasm in the hepatocytes of rats with periodontitis. CONCLUSION: The results obtained suggest that the treatment with αTPN-ßCD improves several oral and blood parameters in rats with experimental periodontitis. In addition, hepatic alterations caused by periodontitis were ameliorated in the rats treated with αTPN-ßCD.

Antimicrobial activity of Phyllanthus amarus Schumach. & Thonn and inhibition of the NorA efflux pump of Staphylococcus aureus by Phyllanthin.

The aim of this study was to evaluate the antimicrobial activity of ethanoic extract of P. amarus (PAEE) and its compound Phyllanthin, as well as, investigate if these natural products could modulate the fluoroquinolone-resistance in S. aureus SA1199-B by way of overexpression of the NorA efflux pump. Microdilution tests were carried out to determine the minimal inhibitory concentration (MIC) of the PAEE or Phyllanthin against several bacterial and yeast strains. To evaluate if PAEE or Phyllanthin were able to act as modulators of the fluoroquinolone-resistance, MICs for Norfloxacin and ethidium bromide were determined in the presence or absence of PAEE or Phyllanthin against S. aureus SA1199-B. PAEE showed antimicrobial activity against Gram-negative strains, meanwhile Phyllanthin was inactive against all strains tested. Addition of PAEE or Phyllanthin, to the growth media at sub-inhibitory concentrations enhanced the activity of the Norfloxacin as well as, Ethidium Bromide, against S. aureus SA1199-B. These results indicate that Phyllanthin is able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. This hypothesis was supported by in silico docking analysis which confirmed that Phyllantin is a NorA ligand. Thus, this compound could be used as a potentiating agent of the Norfloxacin activity in the treatment of infections caused by fluoroquinolone-resistant S. aureus.

Cardiovascular Effect of Diosgenin in Ovariectomized Rats.

Diosgenin is a phytoestrogen and a constituent of Dioscorea. It has several biological effects, and some of them are anti-inflammatory, antidiabetic, antitumor, and vasodilatory. The present study investigated both the vasorelaxing and antioxidant mechanisms of diosgenin in isolated rat aortic rings. Female rats weighing 200-220 g were subjected to sham or OVX operations at 8 weeks of age. Ovariectomy was performed for menopause induction after anesthesia. Diosgenin (10-9 M-3 × 10-4 M) produced a concentration-dependent relaxation in aortic rings precontracted with phenylephrine (1 µM), exhibiting Emax value of 55.34% ± 7.7% (in endothelium-intact rings) and Emax value of 30.30% ± 5.7% (in endothelium-denuded rings). In the endothelium-intact rings, the vasorelaxing effect of diosgenin was reduced by NG-nitro-l-arginine methyl ester (L-NAME) (100 µM), atropine (1 µM), indomethacin (10 µM), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) (10 µM), 4-aminopyridine (1 mM), tetraethylammonium (3 mM), glibenclamide (10 µM), apamin (10 µM), and Tiron (1 µM). Diosgenin (10-5 M) inhibited the contractions induced by cumulative addition of phenylephrine (10-9-10-5 M). The 28-days treatment with diosgenin (50 mg/kg, v.o.) did not imply changes in the myeloperoxidase parameter, but increased significantly, levels of glutathione, superoxide dismutase, and nitric oxide, as well as reduced the concentration of malondialdehyde related to lipid peroxidation. Our results suggest that diosgenin induced relaxation in aortic rings via an endothelium-dependent pathway, which involves the EDRF, the opening of potassium channels and antioxidant action.

Periodontitis causes abnormalities in the liver of rats.

BACKGROUND: Periodontitis not only causes injury to the periodontium, but also damages other tissues such as: articulate, renal, cardiac, and hepatic. The objective of this study was to investigate periodontitis induced alterations in liver function and structure using an experimental model. METHODS: Twenty female rats (Rattus norvegicus) were allocated into two groups: control and periodontitis. Gingival bleeding index and oxidative stress parameters and specific circulating biomarkers were measured. Immunohistochemistry was carried out using alkaline phosphatase (AlkP) staining of the liver. Hepatic tissues, cytokines, and lipid contents were measured. Histopathologic evaluation of the liver was carried out using light and electron microscopy. RESULTS: Liver histopathologic and immunohistochemistry assessment showed increase in steatosis score, and presence of binucleate hepatocytes and positive cells for AlkP in periodontitis versus control group. Ultrastructural evaluation showed significant increase in size and number of lipid droplets (LD), distance between the cisterns of rough endoplasmic reticulum (RER), mitochondria size, foamy cytoplasm, and glycogen accumulation in the liver of the periodontitis group compared with the control group. In addition, plasma levels of AlkP, high-density lipoprotein (HDL), triglycerides, and total cholesterol were also changed. CONCLUSION: Experimental periodontitis caused immunohistochemistry, histopathologic, ultrastructural, oxidative, and biochemical changes in the liver of rats.

Scientific and technological prospection on transdermal formulations and complementary therapies for the treatment of primary dysmenorrhea.

INTRODUCTION: Primary dysmenorrhea (PD) is another term for idiopathic menstrual cramps. Treatments include the use of oral non-steroidal anti-inflammatory drugs (NSAIDs). These drugs have several side effects. The objective of this study was to perform a systematic review on the transdermal administration of drugs and the use of alternative therapies for the treatment of PD. AREAS COVERED: The article bases were Web of Science, PubMed and Sciencedirect and the patent bases were INPI, EPO and WIPO with publications on Primary Dysmenorrhea and associations with Transdermal Administration; Complementary Therapies and Medicinal Plants. 21 articles and 12 patents were analyzed. The results demonstrate the need for alternative therapies for the treatment of PD, with greater effectiveness and lower side effects, mainly in an attempt to reduce the intensity and duration of pain as well as reducing the continuous use of medications. EXPERT OPINION: The study of technological prospection highlighted the relevant importance in seeking new methods for the relief of the symptoms provoked by this condition. The perspectives coexist in the discovery of new natural and biotechnological pharmacological applications, mainly in the development of new devices capable of facilitating and optimizing this form of administration in an attempt to reduce side effects.

Vasorelaxant activity and acute toxicity of the ethanolic extract of Zanthoxylum rhoifolium Lam leaves

The study evaluated the vasorelaxant effect induced by the ethanolic extract of the leaves of Zanthoxylum rhoifolium Lam (EEtOH-Zr/leaves). Wistar rats were treated with the leaf extract containing a single dose of 2,000 mg / kg, v.o. After 14 days, the animals were anesthetized for blood collection and subsequent analysis of the biochemical parameters; they were then euthanized (sodium pentobarbital-100 mg/kg, i.p.) for the removal and morphological analysis of the heart, lung, liver and kidney. The vasorelaxation activity the and vascular reactivity of EEtOH-Zr/leaves were evaluated on artery mesenteric rings isolated from rats. The extract showed no signs of toxicity and no significant difference in the values of the biochemical parameters between the control group and the group of treated animals. In the evaluation of pharmacological activity in the smooth muscle, the EEtOH-Zr/leaves caused vasorelaxant effect on the tonic contraction induced by phenylephrine in mesenteric artery preparations in the presence (pD2=2.17±0.05 µg/mL; Emax=99.8±5.2%) and absence (pD2=2.14±0.05 µg/mL; Emax=95.3±6.4%) of the vascular endothelium. Oral administration of EEtOH-Zr/leaves reduced the contraction induced by the cumulative addition of PHE. It is concluded that the EEtOH-Zr/leaves promote vasorelaxation and reduce vascular reactivity of adrenergic alpha-1 agonist in the mesenteric artery. The results did not show toxic effects of the extract.

Moderate-intensity exercise and renin angiotensin system blockade improve the renovascular hypertension (2K1C)-induced gastric dysmotility in rats.

Actually, arterial hypertension is a major public health concern, which involves the renin angiotensin aldosterone system (RAS), via activation of the angiotensin receptors AT1 and AT2 of the cardiovascular system. Although angiotensin is an important stimulant of the gut permeability to sodium and water, little is known about the effects of arterial hypertension on gut motor behavior. Thus, we evaluated in rats the effect of hypertension induced by two-kidney one-clip (2K1C) model on the gastric motility, as well as the influence of exercise and RAS blockers treatment in such phenomenon. One week after surgery the rats were treated with Aliskiren (50 mg·kg-1, p.o.), Captopril (50 mg·kg-1, p.o.) or Losartan (10 mg·kg-1, p.o). Other group of rats was submitted to swimming with 5% body weight overload. After 4 weeks of physical training or pharmacological treatment, we assessed the gastric retention in all groups (GR) of a liquid test meal, the mean arterial pressure (MAP), the heart rate (HR) and the HR variation (HRV) as well as the in vitro contractility of gastric fundus. Renovascular hypertension increased (p < 0.05) the GR, MAP and HR, a phenomenon prevented by pretreatment with RAS blockers or exercise. The two kidney one-clip Hypertension (2K1C) decreased (p < 0.05) the gastric fundus responsiveness, a phenomenon also prevented by exercise. It conclusion, renovascular hypertension delays the gastric emptying of liquids, a phenomenon involving the activation of RAS, where exercise or blockade with aliskiren, captopril and losartan prevent gastric dysmotility.

A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

Garcinielliptone FC, a polyisoprenylated benzophenone from Platonia insignis Mart., promotes vasorelaxant effect on rat mesenteric artery.

Polyisoprenylated benzophenones represent a group of chemical compounds commonly identified in Clusiaceae species and are responsible for a large amount of biological activities. In this work, the vasorelaxant effect induced by garcinielliptone FC (GFC) isolated from Platonia insignis Mart. (Clusiaceae), a monotype species from Platonia genus, was investigated. GFC promoted an endothelium-independent vasorelaxation on phenylephrine (PHE, 10(-5) mol L(-1))-induced vasoconstriction, but not on KCl (80 mmol L(-1))-induced vasoconstriction, on rat superior mesenteric artery rings. In addition, a concentration-dependent decrease of PHE- or serotonin-induced cumulative concentration-response curves was observed for GFC, and a slight decrease of pD2 value on CaCl2-induced vasoconstriction. In a Ca(2+)-free medium, GFC interfered in calcium mobilisation from PHE (10(-5) mol L(-1))-sensitive intracellular stores. GFC-induced vasorelaxant effect is probably mediated by a dual effect on mobilisation of calcium intracellular stores and attenuation of transmembrane calcium influx.

Antihypertensive and vasorelaxant effects of ethanol extract of stem barks from Zanthoxylum rhoifolium Lam. in rats.

Administration of ethanol extract of stem bark from Z. rhoifolium (EEtOH-ZR) induced hypotension associated with a dual effect in heart rate in normotensive rats. This response was highlighted in spontaneously hypertensive rats (SHR). In rat superior mesenteric artery rings, the cumulative addition of EEtOH-ZR (0.1-750 microg/mL) on a phenylephrine-induced pre-contraction (10(-5) M) promoted a vasorelaxant effect by a concentration-dependent manner and independent of vascular endothelium. A similar effect was obtained on KCl-induced pre-contractions (80 mM). EEtOH-ZR attenuated contractions induced by cumulative addition of CaCl2 (10(-6)-3 x 10(-2) M) in depolarizing medium without Ca2+ only at 500 or 750 microg/mL. Likewise, on S-(-)-Bay K 8644-induced pre-contractions (10(-7) M), the EEtOH-ZR-induced vasorelaxant effect was attenuated. EEtOH-ZR (27, 81, 243 or 500 microg/mL) inhibited contractions induced by cumulative addition of phenylephrine (10(-9) - 10(-5) M) in endothelium-denuded preparations or by a single concentration (10(-5) M) in a Ca(2+)-free medium. The involvement of K+ channels was evaluated by tetraethylammonium (3 mM); the EEtOH-ZR-induced vasorelaxation was not attenuated. Thus, calcium influx blockade through voltage-operated calcium channels (CavL) and inhibition of calcium release from intracellular stores are probably underlying EEtOH-ZR-induced cardiovascular effects.

Citronellal, a monoterpene present in Java citronella oil, attenuates mechanical nociception response in mice.

CONTEXT: Citronellal is a monoterpene present in the oil of many species, including Cymbopogon winterianus Jowitt (Poaceae). OBJECTIVE: The present study investigated the effect of citronellal on inflammatory nociception induced by different stimuli and examined the involvement of the NO-cGMP-ATP-sensitive K⁺ channel pathway. MATERIALS AND METHODS: We used male Swiss mice (n = 6 per group) that were treated intraperitoneally with citronellal (25, 50 or 100 mg/kg) 0.5 h after the subplantar injection of 20 µl of carrageenan (CG; 300 µg/paw), tumor necrosis factor-α (TNF-α; 100 pg/paw), prostaglandin E2 (PGE2; 100 ng/paw) or dopamine (DA; 30 µg/paw). The mechanical nociception was evaluated at 0.5, 1, 2 and 3 h after the injection of the agents, using a digital analgesimeter (von Frey). The effects of citronellal were also evaluated in the presence of L-NAME (30 mg/kg) or glibenclamide (5 mg/kg). RESULTS: At all times, citronellal in all doses inhibited the development of mechanical nociception induced by CG (p < 0.001 and p < 0.01) and TNF-α (p < 0.001, p < 0.01, and p < 0.05). The citronellal was able to increase the pain threshold in the DA test (p < 0.001, p < 0.01, and p < 0.05) and in the PGE2 test at all times (p < 0.001 and p < 0.05). L-NAME and glibenclamide reversed the antinociceptive effects of the citronellal at higher doses in the PGE2 test. DISCUSSION AND CONCLUSION: These data suggest that citronellal attenuated mechanical nociception, mediated in part by the NO-cGMP-ATP-sensitive K⁺ channel pathway.

Calcium channel blockade as a target for the cardiovascular effects induced by the 8 (17), 12E, 14-labdatrien-18-oic acid (labdane-302).

The cardiovascular effects induced by labdane-302, a diterpene isolated from the stems of Xylopia langsdorffianna St. Hill and Tull, were evaluated in male Wistar rats. In normotensive, conscious animals, labdane-302 produced dose-dependent hypotension and tachycardia. These effects were significantly attenuated after pre-treatment with L-NAME (20 mg/kg, i.v.). In isolated mesenteric artery rings, labdane-302 (10(-10)-10(-4)M) elicited concentration-dependent relaxation of phenylephrine-induced contractions (IC50 = 5.4 +/- 1.4 microM). Endothelium removal, and pre-treatment with L-NAME (100 microM) or indomethacin (10 microM) caused significant reductions in sensitivity. Labdane-302 also caused concentration-dependent relaxation in arterial rings pre-contracted with high extracellular KCl (80 mM). In Ca2+-free depolarized preparations, labdane-302 inhibited contractions produced by cumulative increases in extracellular Ca2+ concentration. In GH3 cells, labdane-302 (100 microM) inhibited whole-cell L-type Ca2+ currents by approximately 50%. These results demonstrate that labdane-302 causes hypotension through peripheral vasodilation, mediated in part by NO and PGI2 and by blockade of Ca2+ entry through L-type Ca2+ channels.