Andrea Grabe-Guimarães

Anti-inflammatory and antinociceptive activities of Campomanesia adamantium

ETHNOPHARMACOLOGICAL RELEVANCE Campomanesia species are used in folk medicine as anti-inflammatory, anti-rheumatic, anti-diarrheal and hypocholesterolemic. AIM OF THE STUDY The present study investigated the in vivo anti-inflammatory and antinociceptive properties of ethyl acetate (AE) and aqueous (Aq) extracts from leaves of Campomanesia adamantium and in vitro anti-inflammatory activity of AE and its isolated flavonols, myricitrin and myricetin. MATERIALS AND METHODS The antinociceptive activity of AE and Aq was evaluated using acetic acid-induced writhing and formalin methods. The in vivo anti-inflammatory effect of AE and Aq was evaluated using carrageenan-induced paw oedema in mice. AE, myricitrin and myricetin were evaluated for their abilities to modulate the production of NO, TNF-α and IL-10 in LPS/IFN-γ stimulated J774.A1 macrophages. RESULTS It was found that orally administrated AE and Aq (125 and 250 mg/kg) inhibited carrageenan-induced paw oedema in mice. AE (125 and 250 mg/kg) and Aq (125 mg/kg) reduced the time to licking at the second phase of the formalin method in vivo in mice. AE (250 mg/kg) and Aq (125 mg/kg) also reduced the number of writhes. AE, myricitrin and myricetin inhibited NO (320 μg/mL and 6.25-100 μM, respectively) and TNF-α production by macrophages (320 μg/mL for AE, 100 μM for myricitrin and 25-100 μM for myricetin). AE (160 and 320 μg/mL), myricitrin (50 and 100 μM) and myricetin (25-100 μM) increased IL-10 production by macrophages. CONCLUSIONS The ethyl acetate and aqueous extracts from Campomanesia adamantium showed antinociceptive and anti-inflammatory effects supporting the use of the plant in folk medicine. The results suggest that anti-oedematogenic effect promoted by aqueous extract involves several anti-inflammatory mechanisms of action. The antinociceptive effect shown by aqueous extract can be due to the modulation of release of inflammatory mediators involved in nociception. The anti-inflammatory effects of AE and of its isolated flavonols may be attributed to inhibition of pro-inflammatory cytokines production, TNF-α and NO and to the increased of IL-10 production.

Pharmacological basis for use of Lychnophora trichocarpha in gouty arthritis: Anti-hyperuricemic and anti-inflammatory effects of its extract, fraction and constituents

ETHNOPHARMACOLOGICAL RELEVANCE The ethanolic extract of Lychnophora trichocarpha Spreng. is used in Brazilian folk medicine to treat bruise, pain and inflammatory diseases. AIM OF THE STUDY The present study aimed at investigating whether ethanolic extract of L. trichocarpha, its ethyl acetate fraction and its main bioactive compounds could be useful to treat gouty arthritis by countering hyperuricemia and inflammation. MATERIALS AND METHODS L. trichocarpha ethanolic extract (LTE), ethyl acetate fraction from ethanolic extract (LTA) and isolated compounds were evaluated for urate-lowering activity and liver xanthine oxidase (XOD) inhibition in oxonate-induced hyperuricemic mice. Anti-inflammatory activity in monosodium urate crystal-induced paw oedema, an experimental model of gouty arthritis, was also investigated. RESULTS Crude ethanolic extract and its ethyl acetate fraction showed significant urate-lowering effects. LTE was also able to significantly inhibit liver xantine oxidase (XOD) activity in vivo at the dose of 250mg/kg. Luteolin, apigenin, lupeol, lychnopholide and eremantholide C showed the anti-hyperuricemic activities among tested compounds. Apigenin also showed XOD inhibitory activity in vivo. Luteolin, lychnopholide, lupeol and eremantholide C, in turn, did not shown significant inhibitory activity towards this enzyme, indicating that this mechanism is not likely to be involved in urate-lowering effects of those compounds. LTE, LTA, lupeol, β-sitosterol, lychnopholide, eremantholide, luteolin and apigenin were also found to inhibit monosodium urate crystals-induced paw oedema in mice. CONCLUSIONS Ethanolic extract of Lychnophora trichocarpha and some of its bioactive compounds may be promising agents for the treatment of gouty arthritis since they possesses both anti-hiperuricemic and anti-inflammatory properties.

Profile of wound healing process induced by allantoin

PURPOSE To evaluate and characterize the wound healing process profile induced by allantoin incorporated in soft lotion oil/water emulsion using the planimetric and histological methods. METHODS Female Wistar rats (n=60) were randomly assigned to 3 experimental groups: (C) control group-without treatment; (E) group treated with soft lotion O/W emulsion excipients; (EA) group treated with soft lotion O/W emulsion containing allantoin 5%. The emulsions either containing or not allantoin were topically administered for 14 days and the wound area was evaluated by planimetry and by qualitative and quantitative histological analysis of open wound model. RESULTS The data which were obtained and analyzed innovate by demonstrating, qualitatively and quantitatively, by histological analysis, the profile of healing process induced by allantoin. The results suggest that the wound healing mechanism induced by allantoin occurs via the regulation of inflammatory response and stimulus to fibroblastic proliferation and extracellular matrix synthesis. CONCLUSION This work show, for the first time, the histological wound healing profile induced by allantoin in rats and demonstrated that it is able to ameliorate and fasten the reestablishment of the normal skin.

Anti-inflammatory sesquiterpene lactones from Lychnophora trichocarpha Spreng. (Brazilian Arnica).

The aerial parts of Lychnophora trichocarpha Spreng. (Asteraceae) are used macerated in water or ethanol to treat inflammation, pain, rheumatism, contusions, bruises and insect bites in Brazilian traditional medicine. In this study, anti‐inflammatory activity of ethanol extract from aerial parts of L. trichocarpha and its ethyl acetate fraction was investigated. Sesquiterpene lactones, lychnopholide (Lyc) and eremantholide C (EreC), isolated of ethyl acetate fraction, were also assayed for in vitro and in vivo anti‐inflammatory activity. Topical treatment with ointments containing ethanol extract, its ethyl acetate fraction and sesquiterpene lactones significantly reduced carrageenan‐induced mice paw oedema. In vitro assays demonstrated that Lyc inhibited interferon ‐γ/lipopolysaccharide ‐stimulated nitric oxide (NO) production in J774A.1 macrophages and increased production of IL‐10 anti‐inflammatory cytokine. The reduction of tumor necrosis factor‐α (TNF‐α) production by EreC was accompanied by an increased production of IL‐10 in a concentration‐dependent manner in J774A.1 macrophages. The anti‐inflammatory effect of Lyc seems to involve the inhibition of production of NO and increased production of IL‐10. The mechanism of the effect of EreC on the reduction of carrageenan‐induced paw oedema may be attributed to inhibition of production of TNF‐α and stimulation of IL‐10 production. The results corroborate the use of ethanol extract from Lychnophora trichocarpha in folk medicine for anti‐inflammatory action and indicate that the topical route is suitable for use. Copyright

Prolonged cardioprotective effect of pyridostigmine encapsulated in liposomes

AIMS The purpose of the present work was to investigate the ability of pyridostigmine encapsulated in long-circulating liposomes, to protect against ECG (electrocardiogram) alterations induced by sympathetic stimulation in rats. MAIN METHODS The encapsulation of pyridostigmine was carried out by freeze-thaw and extrusion. Blood pressure and ECG (limb lead II) were monitored in anaesthetized male Wistar rats. The formulation containing pyridostigmine was intravenously administrated in 0.1, 0.3 and 1.0mg/kg doses, and sympathetic stimulation was conducted by administration of 1 or 3 microg of noradrenaline (NA) after 1, 2, 4 or 6h. The obtained cardiovascular parameters were compared to animals that received intravenous injection of pyridostigmine in free form or saline. KEY FINDINGS After saline, NA induced a significant increase in QT interval (22.3% after 3.0 microg). Previous administration of free pyridostigmine significantly prevented the increase of QT interval after sympathetic stimulation and the most prominent effect was observed after 1h for the dose of 0.3mg/kg (6.8% after 3.0 microg of NA) and was no longer observed after 2h of the treatment. On the other hand, the maximum effect of pyridostigmine in liposomal formulation preventing QT interval increase was observed 2h after treatment (9.7% after 3.0 microg of NA) and was still present until 6h when 1mg/kg was previous administrated. SIGNIFICANCE The results of the present study, beyond to confirm the cardioprotective action of pyridostigmine, suggest that liposomal pyridostigmine may be a potential therapeutic alternative to prevent cardiovascular disturbances resulting from sympathetic hyperactivity.

Reduced cardiovascular alterations of tartar emetic administered in long-circulating liposomes in rats.

Trivalent antimonial drugs, including tartar emetic (TA), are known to induce important cardiotoxicity observed by electrocardiographic abnormalities. Liposome encapsulation was found to reduce the overall acute toxicity of TA. The present work investigated the cardiovascular parameters alterations of rats submitted to the treatment with free and encapsulated TA in long-circulating liposomes. Liposomes were made using lipids DSPC, DSPE-PEG and cholesterol. The cardiovascular signals, electrocardiogram (ECG) and arterial blood pressure (AP), were recorded from anaesthetized Wistar rats after intravenous (IV) administration of a single specially high dose (17 mg/kg) of TA in liposomes and in free form. The IV administration of TA solution caused significant increase of QT interval of ECG and significant reduction of AP when compared to the control group. These alterations were not observed when liposomes TA were administered and the profile of ECG and AP data was quite similar to the control groups. In conclusion, a liposomal formulation of TA showed a reduced cardiotoxic profile for TA when compared to the free form.

Biodegradable Polymeric Nanocapsules Prevent Cardiotoxicity of Anti-Trypanosomal Lychnopholide

Chagas disease is a neglected parasitic disease caused by the protozoan Trypanosoma cruzi. New antitrypanosomal options are desirable to prevent complications, including a high rate of cardiomyopathy. Recently, a natural substance, lychnopholide, has shown therapeutic potential, especially when encapsulated in biodegradable polymeric nanocapsules. However, little is known regarding possible adverse effects of lychnopholide. Here we show that repeated-dose intravenous administration of free lychnopholide (2.0 mg/kg/day) for 20 days caused cardiopathy and mortality in healthy C57BL/6 mice. Echocardiography revealed concentric left ventricular hypertrophy with preserved ejection fraction, diastolic dysfunction and chamber dilatation at end-stage. Single cardiomyocytes presented altered contractility and Ca2+ handling, with spontaneous Ca2+ waves in diastole. Acute in vitro lychnopholide application on cardiomyocytes from healthy mice also induced Ca2+ handling alterations with abnormal RyR2-mediated diastolic Ca2+ release. Strikingly, the encapsulation of lychnopholide prevented the cardiac alterations induced in vivo by the free form repeated doses. Nanocapsules alone had no adverse cardiac effects. Altogether, our data establish lychnopholide presented in nanocapsule form more firmly as a promising new drug candidate to cure Chagas disease with minimal cardiotoxicity. Our study also highlights the potential of nanotechnology not only to improve the efficacy of a drug but also to protect against its adverse effects.

Development and characterization of multilamellar liposomes containing pyridostigmine

Abstract Pyridostigmine has cardioprotective activity in both free and liposomal forms. This study aimed to develop and characterize liposomal formulations of pyridostigmine. For this, a spectrophotometric ultraviolet (UV) analytical method, at 270 nm, was developed and validated to quantify liposomal pyridostigmine. The method was linear in ranges from 0.02 to 0.09 mg/mL. The accuracy of this method was determined intra- and inter-day; the results of coefficient of variation were of 1.73–2.72% and 0.32–2.32%, respectively. The accuracy ranged between 99.45% and 101.12%. The method has not changed by influence of liposomal matrix and demonstrated being able to quantify pyridostigmine in liposomes. Two liposomal multilamellar formulations were developed: a constituted by dystearoyl-phosphatidylcholine (DSPC) and cholesterol (CHOL) other by dioleil-phosphatidylcholine (DOPC) and CHOL. The encapsulation efficiency was determined as 23.4% and 15.4%, respectively. Analyses of size and release of pyridostigmine from the formulations were made and the results showed that the formulations are viable for future studies in vivo.

Effect of pyridostigmine on in vivo and in vitro respiratory muscle of mdx mice

The current work was conducted to verify the contribution of neuromuscular transmission defects at the neuromuscular junction to Duchenne Muscular Dystrophy disease progression and respiratory dysfunction. We tested pyridostigmine and pyridostigmine encapsulated in liposomes (liposomal PYR), an acetylcholinesterase inhibitor to improve muscular contraction on respiratory muscle function in mdx mice at different ages. We evaluated in vivo with the whole-body plethysmography, the ventilatory response to hypercapnia, and measured in vitro diaphragm strength in each group. Compared to C57BL10 mice, only 17 and 22 month-old mdx presented blunted ventilatory response, under normocapnia and hypercapnia. Free pyridostigmine (1mg/kg) was toxic to mdx mice, unlike liposomal PYR, which did not show any side effect, confirming that the encapsulation in liposomes is effective in reducing the toxic effects of this drug. Treatment with liposomal PYR, either acute or chronic, did not show any beneficial effect on respiratory function of this DMD experimental model. The encapsulation in liposomes is effective to abolish toxic effects of drugs.

Swim training attenuates the adverse remodeling of LV structural and mechanical properties in the early compensated phase of hypertension

Aim: Investigate to what extent low‐intensity swim training for six weeks counterbalances the adverse remodeling due to the advance of pathological hypertrophy in the left ventricle (LV) structural and mechanical properties in the early compensated phase of hypertension in male SHR. Main methods: Four‐month‐old male SHR and Wistar rats were randomly divided into Sed (sedentary) and Ex (exercised) groups. The exercised rats were submitted to a swimming protocol (1 h/day, 5 times/week, no additional load) for six weeks. LV tissue and isolated myocytes were used to assess structural and mechanical properties. Myocytes were stimulted at frequencies (F) of 1 and 3 Hz at 37 °C. Key findings: Exercised SHR showed improvement in cardiovascular parameters compared to sedentary SHR (mean arterial pressure: 13.22%; resting HR: 14.28.%). About structural and mechanical properties, swim training induced a decrease in LV myocyte thickness (10.85%), number of inflammatory cells (21.24%); collagen type III (74.23%) and type I (85.6%) fiber areas; amplitude of single myocyte shortening (47% to F1 and 28.46% to F3), timecourses of shortening (16.5% to F1 and 7.55% to F3) and relaxation (15.31% to F3) compared to sedentary SHR. Significance: Six weeks of swim training attenuates the adverse remodeling of LV structural and mechanical properties in the early compensated phase of hypertension in male SHR.