Analgesic and anti-inflammatory activities of ethyl acetate and butanol fractions from Vitex polygama hydroalcoholic leaf extract.

This study evaluated the analgesic and anti-inflammatory activities of Vitex polygama. Ethyl acetate and butanol fractions (10-30 mg/kg), obtained from the hydroalcoholic leaf extract, showed an antinociceptive effect in the acetic acid-induced abdominal writhing test, formalin test and modified hot plate test in mice, indicating a peripheral anti-inflammatory action. Ethyl acetate and butanol fractions were effective in inhibiting nitric oxide and TNF-α production, respectively, in RAW 264.7 macrophages. Both fractions (10-30 mg/kg) showed an acute analgesic effect in mice with vincristine-induced neuropathic pain exposed to a thermal stimulus. Through ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-UV-MS/MS) it was possible to identify seven major compounds: isoorientin, orientin, vitexin, isovitexin, O-p-hydroxybenzoyl orientin, O-caffeoyl-orientin, and di-caffeoylquinic acid. Orientin and isoorientin were isolated from ethyl acetate fraction and had their identity confirmed by nuclear magnetic resonance (NMR). Glucosyl flavones appear to be the main metabolites responsible for the anti-inflammatory and analgesic activities observed for V. polygama.

A new bioinspired peptide on defensin from C. annuum fruits: Antimicrobial activity, mechanisms of action and therapeutical potential.

BACKGROUND: Antimicrobial peptides, natural or synthetic, appear as promising molecules for antimicrobial therapy because of their both broad antimicrobial activity and mechanism of action. Herein, we determine the anti-Candida and antimycobacterial activities, mechanism of action on yeasts, and cytotoxicity on mammalian cells in the presence of the bioinspired peptide CaDef2.1G27-K44. METHODS: CaDef2.1G27-K44 was designed to attain the following criteria: high positive net charge; low molecular weight (<3000 Da); Boman index ≤2.5; and total hydrophobic ratio ≥ 40%. The mechanism of action was studied by growth inhibition, plasma membrane permeabilization, ROS induction, mitochondrial functionality, and metacaspase activity assays. The cytotoxicity on macrophages, monocytes, and erythrocytes were also determined. RESULTS: CaDef2.1G27-K44 showed inhibitory activity against Candida spp. with MIC100 values ranging from 25 to 50 µM and the standard and clinical isolate of Mycobacterium tuberculosis with MIC50 of 33.2 and 55.4 µM, respectively. We demonstrate that CaDef2.1G27-K44 is active against yeasts at different salt concentrations, induced morphological alterations, caused membrane permeabilization, increased ROS, causes loss of mitochondrial functionality, and activation of metacaspases. CaDef2.1G27-K44 has low cytotoxicity against mammalian cells. CONCLUSIONS: The results obtained showed that CaDef2.1G27-K44 has great antimicrobial activity against Candida spp. and M. tuberculosis with low toxicity to host cells. For Candida spp., the treatment with CaDef2.1G27-K44 induces a process of regulated cell death with apoptosis-like features. GENERAL SIGNIFICANCE: We show a new AMP bioinspired with physicochemical characteristics important for selectivity and antimicrobial activity, which is a promising candidate for drug development, mainly to control Candida infections.

Antimycobacterial and anti-inflammatory activities of thiourea derivatives focusing on treatment approaches for severe pulmonary tuberculosis.

Tuberculosis (TB) remains a serious public health problem and one of the main concern is the emergence of multidrug-resistant and extensively resistant TB. Hyper-reactive patients develop inflammatory necrotic lung lesions that aggravate the pathology and facilitate transmission of mycobacteria. Treatment of severe TB is a major clinical challenge that has few effective solutions and patients face a poor prognosis, years of treatment and different adverse drug reactions. In this work, fifteen novel and thirty-one unusual thiourea derivatives were synthesized and evaluated in vitro for their antimycobacterial and anti-inflammatory potential and, in silico for ADMET parameters and for structure-activity relationship (SAR). Thioureas derivatives 10, 15, 16, 28 and 29 that had shown low cytotoxicity and high activities were selected for further investigation, after SAR study. These five thioureas derivatives inhibited Mtb H37Rv growth in bacterial culture and in infected macrophages, highlighting thiourea derivative 28 (MIC50 2.0 ± 1.1 and 2.3 ± 1.1 µM, respectively). Moreover, these compounds were active against the hypervirulent clinical Mtb strain M299, in bacterial culture, especially 16, 28 and 29, and in extracellular clumps, highlighting 29, with MIC50 5.6 ± 1.2 µM. Regarding inflammation, they inhibited NO through the suppression of iNOS expression, and also inhibited the production of TNF-α and IL-1ß. In silico studies were carried out suggesting that these five compounds could be administered by oral route and have low toxicological effects when compared to rifampicin. In conclusion, our data show that, at least, thiourea derivatives 16, 28 and 29 are promising antimycobacterial and anti-inflammatory agents, and candidates for further prospective studies aiming new anti-TB drugs, that can be used on a dual approach for the treatment of severe TB cases associated with exacerbated inflammation.

Evaluation of the chemical composition and antioxidant activity of extracts and fractions of Ocotea notata (Ness) Mez (Lauraceae).

The specie Ocotea notata (Nees & Mart). Mez is a tree with 5 meters high, that can be found in restinga regions in the Brazilian coast. This study describes a phytochemical investigation, total phenolic content and the antioxidant activity of extracts and fractions by DPPH and ORAC. Phenolic content revealed equivalent concentration between evaluated samples, similar to found in the leave extract (66.4 mEq GA/g). By DPPH, extracts and fractions showed effective concentration (EC50) lower than the standards Ginkgo biloba 761® (23.60 ± 0.64 µg/ml) and quercetin (6.06 ± 0, 92 µg/mL); for the ORAC method, ethyl acetate partition showed a value of 2.06 mmol Trolox equivalent g-1 better than obtained in Ginkgo biloba (1.03 ± 0.25 mmol.Trolox equivalent g-1. The butanol partition (0.52 mmol.Trolox equivalent g-1) and the aqueous residue (0.74 mmol Trolox equivalent g-1) have a lesser ORAC potential than ethyl acetate partition. The butanolic partition, investigated by LC-DAD-MS/MS and QTOF-MS, revealed six major compounds: miquelianin (1), isoquercitrin (2), quercitrin (3), kaempferol-3-O-pentose (4), afzelin (5) and isorhamnetin-glucuronide (6).

Suppressive effects of Vochysia divergens aqueous leaf extract and its 5-methoxyflavone on murine macrophages and lymphocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Vochysia divergens Pohl (Vochysiaceae), popularly known as "Cambará", is a tree that is resistant to the seasonal floods in the Pantanal, and usually found in monodominant stands called "Cambarazal". The inhabitants of the Pantanal exploit this tree for medicinal uses. Infusions and decoctions of its leaves are taken as teas, particularly for the treatment of asthma, flu and diarrhea, according to the local tradition transmitted empirically through the generations. AIM OF THE STUDY: To evaluate the beneficial health effects related to the ethnomedicinal uses of V. divergens (Vd) by using biomonitored fractionation of an aqueous leaf extract. MATERIALS AND METHODS: The aqueous leaf extract was obtained by decoction, and then the extract was fractionated by a combination of separation techniques including precipitation, organic partition and chromatography. Chromatographic analyses of the active samples were carried out using HPLC-DAD-MS. Flavonoid 1 was isolated from the n-BuOH fraction through classic chromatographic techniques. The inhibitory effects and cytotoxicity of the Vd extract, fractions and flavonoid 1 on NO and TNF-α production were assessed in LPS-stimulated RAW 264.7 macrophage cultures. Additionally, suppression on the proliferation of BALB/c lymphocytes was estimated by [3H] thymidine incorporation. The antioxidant activity of the samples was verified by SNP and DPPH assays and the suppression of the iNOS protein expression was evaluated through Western blotting. RESULTS: The HPLC-DAD-MS analysis of the Vd extract led to the identification of 5-methoxyluteolin-7-O-ß-glucopyranoside (2), rutin (4) and the tannin galloyl-HHDP-glucopyranoside (3), besides the main flavonoid 3',5-dimethoxyluteolin-7-O-ß-glucopyranoside (1), which was biologically evaluated in comparison with luteolin aglycone. The Vd extract, n-BuOH fraction and flavonoid 1 inhibited NO and TNF-α production by LPS-stimulated macrophages. The reduction of NO levels was mediated mainly by suppression of the iNOS expression. In addition, both the Vd extract (IC50 13.6 µg/mL) and flavonoid 1 (IC50 19.8 µg/mL; 41.6 µM) strongly inhibited stimulated lymphocyte proliferation when compared to the immunosuppressive agent cyclosporin A (IC50 43.8 µg/mL; 36.4 µM). The Vd extract also showed a scavenging activity toward DPPH and NO free radicals. This is the first report describing the immunomodulatory potential of V. divergens and its major flavonoid (1). CONCLUSION: Our findings showed that the aqueous leaf extract of V. divergens and its flavonoid reduced the production of excessive pro-inflammatory markers, collaborating with the Pantanal folk medicinal tradition that recommends the tea of cambará leaves for both asthma and flu. In addition, this study contributes to the knowledge of the pharmacological properties of 5-methoxy flavones, a poorly investigated subclass of flavonoids.

Biological activities and phytochemical profile of Passiflora mucronata from the Brazilian restinga

ABSTRACT In general, Passiflora species have been reported for their folk medicinal use as sedative and anti-inflammatory. However, P. caerulea has already been reported to treat pulmonary diseases. Severe pulmonary tuberculosis, generally caused by Mycobacterium tuberculosis strains resistant to multiple drugs, can lead to deleterious inflammation and high mortality, encouraging new approaches in drug discovery. Thus, the aim of this work was to evaluate the Passiflora mucronata Lam., Passifloraceae, potential for tuberculosis treatment. Specifically, related to antimycobacterial activity and anti-inflammatory related effects (based on inhibition of nitric oxide, tumor necrosis factor-alpha production and antioxidant potential), as well as the chemical profile of P. mucronata. High performance liquid chromatography coupled with diode-array ultraviolet and mass spectrometer analyses of crude hydroalcoholic extract and ethyl acetate fraction showed the presence of flavonoids. Ethyl acetate fraction showed to be as antioxidant as Ginkgo biloba standard extract with EC50 of 14.61 ± 1.25 µg/ml. One major flavonoid isolated from ethyl acetate fraction was characterized as isoorientin. The hexane fraction and its main isolated compound, the triterpene β-amyrin, exhibited significant growth inhibitory activity against Mycobacterium bovis BCG (MIC50 1.61 ± 1.43 and 3.93 ± 1.05 µg/ml, respectively). In addition, Passiflora mucronata samples, specially hexane and dichloromethane fractions, as well as pure β-amyrin, showed a dose-related inhibition of lipopolysaccharide (LPS)-induced nitric oxide production. In conclusion, Passiflora mucronata presented relevant biological potential and should be considered for further studies using in vivo pulmonary tuberculosis model.

The Hydroalcoholic Extract of Leaves of Mandevilla moricandiana Induces NO-Mediated Vascular Relaxation.

Natural products extracted from plants represent a valuable source of new bioactive substances. Many studies describe the potential of plant products for the treatment of cardiovascular diseases. Species of the Mandevilla genus have been studied for their biological activities, mainly as antioxidant, anti-inflammatory, and vasorelaxant. However, the phytochemical and pharmacological profiles of Mandevilla moricandiana have not been investigated yet. The aim of this study was to evaluate the vasodilator effect of the hydroalcoholic extract of the leaves of M. moricandiana, as well as its chemical profile. Chemical analysis and quantification of major compounds were performed by HPLC analysis. Total flavonoid content was quantified based on rutin equivalents, and major compounds were identified based on HPLC-DAD-MS analysis. M. moricandiana leaf extract-induced vasodilation was investigated in rat aortic rings precontracted with phenylephrine. The total flavonoids were quantified as 3.25 ± 0.11 % w/w of the hydroalcoholic leaf extract, and HPLC-DAD-MS allowed for the identification of luteolin and quercetin glycosides. The maximal relaxant effect of the hydroalcoholic leaf extract was 86.07 ± 1.68 % at a concentration of 30 µg/mL (p < 0.05; n = 6). The concentration of hydroalcoholic extract of the leaves of M. moricandiana necessary to reduce phenylephrine-induced contractions of the endothelium-intact aorta by 50 % was 0.82 ± 0.10 µg/mL. M. moricandiana leaf extract-induced vasodilation was abolished in aortas pretreated with NG-nitro-L-arginine methyl ester and 1H-[1,2,4]oxadiazolo-[4,3-α]quinoxalin-1-one. In addition, diphenhydramine partially inhibited the effect of the hydroalcoholic extract of the leaves of M. moricandiana. Thus, M. moricandiana-induced relaxation depends on the endothelium and on the activation of the nitric oxide/cyclic GMP pathway, with the involvement of endothelial histamine H1 receptors. Luteolin and quercetin glycosides seem to contribute to the extract activity.

Ultraviolet-B radiation effects on phenolic profile and flavonoid content of Kalanchoe pinnata.

Ultraviolet-B radiation is an important abiotic factor that can stimulate the production of secondary metabolites, including polyphenolic compounds. Kalanchoe pinnata (Crassulaceae) is a medicinal plant popularly used in Brazil for treating wounds and inflammation. This species is rich in phenolic compounds, which could account for some of its biological activities, including antileishmanial, antihypertensive and antibacterial properties. We investigated the effects of supplemental UV-B radiation on the phenolic profile, antioxidant activity and total flavonoid content of leaves of K. pinnata. Plants were grown under white light (W - control) and supplemental UV-B radiation (W+UVB). Supplemental UV-B radiation enhanced the total flavonoid content of the leaf extracts, without affecting the antioxidant activity or yield of extracts. Analysis by TLC and HPLC of W and W+UVB leaf extracts revealed quantitative and qualitative differences in their phenolic profiles. W+UVB extracts contained a higher diversity of phenolic compounds and a larger amount of quercitrin, an important bioactive flavonoid of this species. This is the first report of the use of ImageJ® program to analyze a TLC visualized by spraying with NP-PEG reagent. UV-B radiation is proposed as a supplemental light source in K. pinnata cultivation in order to improve its flavonoid composition.

Sesquiterpenes from the Brazilian red alga Laurencia dendroidea J. Agardh.

Two new chamigrane sesquiterpenes 1-2 and three known compounds 3-5 were isolated from a lipophilic extract of the red alga Laurencia dendroidea collected from the Southeastern Brazilian coast. Dendroidone (1) and dendroidiol (2) were isolated from samples collected at Biscaia Inlet, Angra dos Reis, Rio de Janeiro and at Manguinhos Beach, Serra, Espírito Santo, respectively. Debromoelatol (3), obtusane (4) and (1S*,2S*,3S*,5S*,8S*,9S*)-2,3,5,9-tetramethyltricyclo[6.3.0.0¹·5]undecan-2-ol (5) were obtained from specimens collected at Vermelha Beach, Parati, Rio de Janeiro. The structures of new compounds were elucidated by extensive NMR (¹H-, ¹³C-, COSY, HSQC, HMBC and NOESY) and high resolution mass spectrometry analysis. Additionally, the absolute configuration of compound 2 was assigned by X-ray analysis. Full spectroscopic data is described for the first time for compound 3. Anti-inflammatory and antimycobacterial activities of compounds 2-5 were evaluated. Compounds 3-5 inhibited the release of inflammatory mediator NO while TNF-α levels were only affected by 3. All compounds tested displayed moderate antimycobacterial action.

Antimycobacterial activity and alkaloid prospection of Psychotria species (Rubiaceae) from the Brazilian Atlantic Rainforest.

Ten Psychotria species were collected in two fragments of Atlantic Forest in Rio de Janeiro: Psychotria pubigera (P1A and B), P. ruelliifolia (P2), P. suterela (P3), P. stachyoides (P4), P. capitata (P5), P. glaziovii (P6), P. leiocarpa (P7), P. nuda (P8), P. racemosa (P9) and P. vellosiana (P10). Ethanol extracts of these species were evaluated for their antimycobacterial activity, in an attempt to find new antituberculosis agents. Psychotria pubigera (P1A), P. ruelliifolia (P2) and P. stachyoides (P4) were the most active against Mycobacterium. The anti-inflammatory potential of these extracts was also evaluated in vitro to learn if they inhibit nitric oxide (NO) production in macrophages and if they have free-radical scavenging properties, because inflammation is a severe problem caused by tuberculosis, especially when the infection is from M. bovis or M. tuberculosis. Psychotria suterela (P3), P. stachyoides (P4) and P. capitata (P5) were the most active in inhibiting macrophage NO production but they were not the most antioxidant species. This suggests that NO inhibitory activity is not due to the scavenging of NO generated but due to a specific inhibition of iNOS activity or expression. In addition, cytotoxicity was tested in the macrophages (the host cells of the Mycobacterium) and it was verified that the extracts selectively killed the bacteria and not the host cells. When analyzing antimycobacterial, cytotoxicity and NO inhibitory activities in combination, P. stachyoides (P4) was the most promising anti-TB extract tested. Further, indol alkaloids were detected in P. suterela and P. nuda, and 5,6-dihydro-ß-carboline alkaloids in all of the species studied, with the highest amounts found in P. capitata and P. racemosa.