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Ethnopharmacological relevance Plantago australis is a popular plant found to be widely spread in Latin America. In folk medicine, the seeds and leaves are used mainly for anti-inflammatory, wound healing, among others. The verbascoside, a phenolic glycoside, is an active chemical component described in this species of plant, which has antioxidant, anti-inflammatory and healing effects. Purpose The aim of the present study was to evaluate whether P. australis hydroethanolic extract (PAHE) standardized in verbascoside could promote wound healing associated with anti-inflammatory action within both in vitro and in vivo models. Methods For the wound healing activity, we used a Scratch Test, an assay capable of evaluating the migratory ability of keratinocyte cells (HaCat) in vitro and thereby confirming the activity in rats. For the anti-inflammatory activity, the inflammation was induced with LPS in microglial murine cells (N9). Inflammatory mediators (IL-6, IL-10, IL-12p70, INFγ, MCP-1 and TNFα) were measured and the activity of superoxide dismutase (SOD), catalase (CAT), and mitochondrial membrane potential were evaluated. In addition, using paw edema induced by carrageenan in rats, the anti-inflammatory activity in vivo was analyzed. Results The PAHE and verbascoside, induced a significant increase in migration of keratinocytes, at all concentrations tested when compared to the negative control. The wound healing activity in vivo showed that the PAHE accelerated the process. The treatments with PAHE and verbascoside induce increases in the antioxidants enzymes, suggesting a possible activation of these enzymes. However, this did not result in an increase in the expression of inflammatory mediators in microglial cells. In LPS activated cells the verbascoside displayed a significant reduction of TNFα, IL-6, IL-12p70, MCP-1 and INFγ, while the PAHE only displayed statistically significant reduction in TNFα. Interestingly, both the compounds could reduce the oxidative parameters in N9 cells activated by LPS. Additionally, pretreatment with PAHE inhibited the paw edema in rats. Conclusion The results suggest that PAHE has wound healing activity, improving cells migration and, as well as was able to reverse the oxidation effect in LPS-activated N9 cells. The wound-healing and anti-inflammatory activities of PAHE were confirmed in vivo. In addition, the presence of verbascoside can be related to PAHE effects, since this compound was capable of increase keratinocytes migration and inhibiting inflammation mediators.

Animal models show that cocaine sensitization, a behavioral marker of addiction, is more significant in intact gonadal female than male rats and ovariectomy suppress this behavior in female rats. However, few studies explore changes in neurotransmission related to this phenomenon. Here we investigated the in vivo changes on GABA, glutamate, and taurine levels in the medial prefrontal cortex (mPFC) of gonadal intact or ovariectomized female rats after a cocaine challenge administration. Adult female rats were bilaterally ovariectomized (OVX), or sham-operated (SHAM) and randomly assigned to control (CTR), acute (ACT), or repeated (RPT) cocaine administration groups. In the challenge day, after eight days of daily cocaine (15 mg/kg) or saline administration and ten days of washout and stereotaxic surgery, RPT and ACT groups received cocaine, and the CTR group received saline. Horizontal locomotion was monitored concomitantly with microdialysate collection to determine extracellular GABA, glutamate, and taurine levels. Hormonal determination in blood samples confirmed the lower hormonal status of the OVX. Cocaine sensitization occurred in SHAM-RPT female rats after the challenge administration. Non-sensitized OVX-RPT rats showed a peak of GABA at 30 min after cocaine administration, with no change on glutamate and taurine levels. Therefore, elevated GABA levels in the mPFC and lower serum estrogen levels abolish cocaine sensitization behavior in ovariectomized female rats. We discuss some possible implications of these finding for future models of cocaine sensitization research lighting in the female hormonal influence.

Ethnopharmacological relevance Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. Aims of the study The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. Materials and methods Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (22) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. Results Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. Conclusion The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.

By the classical definition naphthenic acids (NAs) are a complex mixture of monocarboxylic acids containing one or more alkyl-substituted alicyclic rings or only aliphatic carbon chains. Based on the great interest of the oil industry in the elucidation and characterization of naphthenic acids in petrochemical samples, and the lack of efficient methodologies for the total characterization of these compounds, the goal of this study is to compare three techniques (GC/qMS, ESI-MS/MS and GC × GC/TOF-MS) for the analysis of two technical mixtures of NAs. The results presented here demonstrate the applicability of these techniques and the main advantages of each one. GC/qMS is widely used in the naphthenic acid analysis and still presents a good performance; however the ESI-MS-MS allowed a very quick analysis that provided a general profile of each sample, very useful in preliminary classification of petrochemical mixtures, while GC × GC/TOF-MS delivered a thorough evaluation of the different components of each sample. Technical mixtures analyzed showed different profiles confirmed through the three techniques applied. The mixture SA (Sigma Aldrich) presented a predominance of aliphatic acids (Z = 0) while the mixture MN (Miracema-Nuodex) was composed mainly of naphthenic monocyclic acids.

Ethnopharmacological relevance The leaves of Syzygium cumini (L.) or Skeels (Myrtaceae) are widely used in Brazilian folk medicine to treat diabetes. Aim of the study The present study evaluated the functional capacity, biochemical parameters, oxidative stress and DNA damage from eight weeks of intervention with a crude hydroalcoholic extract of S. cumini leaves (EBH) and continuous aerobic training (TAC) in diabetic (D) rats. Materials and methods A hydroalcoholic (50%) extract was prepared by ultrasound and phytochemical parameters (total phenols, total tannins and myricetin content) were analyzed. Thirty-seven male Wistar rats were divided into five groups: normoglycemic controls (CONT), diabetic controls (D-CONT), diabetics treated with extract (D+EBH), trained diabetic (D+TAC) and diabetics treated with extract and trained (D+EBH+TAC). Functional capacity was assessed with a maximum exercise capacity test; biochemical parameters with enzymatic kits; oxidative stress by superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and oxidized dichlorofluorescein (DCF), and the DNA damage by the comet assay. Results The D+TAC and D+EBH+TAC groups showed better functional capacity at the end of interventions. The D+EBH group showed glucose and triglyceride reduction, lowest DNA damage index in the blood, liver, kidney, heart, lung and gastrocnemius muscle, improved SOD levels in the liver, kidney and lung, improved CAT levels in the kidney and lower lipid peroxidation in all tissues studied, compared to the D-CONT group. The exercise (D+TAC) was effective in reducing triglycerides, improving SOD levels in the lung, reducing lipid peroxidation in all tissues studied and reducing the DCF oxidation in the kidney, in addition to protecting against DNA damage in the blood and heart. However, the additive effect of the intervention protocols when combined (EBH+TAC) was observed only in improving the gastrocnemius SOD levels. The phytochemical analyses showed a high content of phenols and the presence of myricetin glycosides. Conclusion The findings in this study suggest a crude hydroalcoholic extract of S. cumini leaves has potential hypoglycemic, hypolipidemic and protective properties acting against oxidative stress and against DNA damage, probably due to its phenols and myricetin glycoside content and the antioxidant properties of these constituents. Moreover, exercise was suggested to have beneficial effects on diabetes, improving functional capacity, ameliorating blood triglyceride control and decreasing lipid peroxidation, but with no effects on ameliorating blood glucose levels. The association of intervention protocols presented an additive effect on the antioxidant SOD activity in the muscle cells of diabetic rats.

Ethnopharmacological relevance Bauhinia platypetala Burch. is a traditionally used Brazilian medicinal plant, although no evidence in the literature substantiates the safety of its use. Aim of the study The aim of this study was to investigate the safety of the ethanolic extract and the ethereal fraction of B. platypetala leaves. Materials and methods The identification of chemical compounds from the B. platypetala ethanolic extract and its ethereal fraction was performed by GC/MS and ESI-MS/MS. The plant’s toxicological, cytotoxic, mutagenic and genotoxic properties were determined in Saccharomyces cerevisiae strains and V79 cell culture by survival assays and comet assay. Results The major compound identified in the B. platypetala ethanolic extract is palmitic acid, kaempferitirin and quercitrin, while the B. platypetala ethereal fraction was found to be rich in phytol, gamma-sitosterol and vitamin E. Moreover, the results indicated that the B. platypetala ethanolic extract has an anti-oxidative effect against H 2 O 2 in yeast. In addition, the B. platypetala ethanolic extract did not induce mutagenic effects on the S. cerevisiae N123 strain, but the ethereal fraction of B. platypetala at higher concentrations (250–500 μg/mL) induced cytotoxicity and mutagenicity. A slight cytotoxic effect was observed in mammalian V79 cells; however, both the B. platypetala ethanolic extract and its ethereal fraction were able to induce DNA strand breaks in V79 cells, as detected by the alkaline comet assay. Conclusion The B. platypetala ethanolic extract has antioxidant action and showed absence of mutagenic effects in yeast S. cerevisiae. On the other hand B. platypetala ethereal fraction is mutagenic and does not show antioxidant activity in yeast. In mammalian cells B. platypetala ethanolic extract and it's ethereal fraction induce cyotoxic and genotoxic action.

Platonia insignis Mart., a native species of the Brazilian Amazon more commonly known as bacuri, is a member of the Clusiaceae family. In this study, we evaluated the chemical composition and the antioxidant and toxicity activities of the dichloromethane and ethyl acetate fractions from P. insignis seed ethanolic extract using different experimental models. Our results demonstrate in vitro antioxidant effects, by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt and 1,1-diphenyl-2-picryl-hydrazyl assays, as well as in vivo effects in antioxidant-defective Saccharomyces cerevisiae strains to both fractions. Toxicity was evaluated against the micro-crustaceous Artemia salina Leach. and promastigote Leishmania amazonensis. The dichloromethane fraction was the most active fraction evaluated on A. salina and promastigote L. amazonensis (IC(50) = 24.89 μg/mL and 2.84 μg/mL, respectively). In addition, a slight cytotoxicity was observed in mammalian V79 cells using ethyl acetate and dichloromethane fractions with MTT assays. Both fractions displayed genotoxicity up to 25 μg/mL (dichloromethane) and 10 μg/mL (ethyl acetate) in V79 cells, as evaluated by the alkaline comet assay. Thus, in this study, we demonstrate for the first time that ethyl acetate and dichloromethane fractions from P. insignis seeds display antioxidant effects, a toxic effect against A. salina and L. amazonensis and induce genotoxicity in V79 mammalian cells. The observed activities can be attributed to the phenolic compounds present in these fractions and to the presence of xanthones (alpha- and gamma-mangostin).

This paper describes a comparative study of extraction methods of terpenes (terpenic alcohols and phytosterols), fatty acids and Vitamin E from leaves of Piper gaudichaudianum Kunth. The analysis of extracts was done by gas chromatography with mass spectrometric detection. The identification and quantification was made by co-injections of the extract with certified standards. The use of pressurized liquid extraction (PLE; Dionex trade name: ASE, for accelerated solvent extraction) decrease significantly the total time of extraction, the amount of solvent and the manipulation of sample and solvents in comparison with soxhlet (SE) and ultrasound-assisted (USE). In addition, PLE was more effective for the extractions of terpenes (terpenic alcohols and phytosterols), fatty acids and Vitamin E.

Piper gaudichaudianum Kunth is used in popular medicine as anti-inflamatory and against liver disorders. One of the most studied components of the plant is the essential oil for which chemical analysis revealed (E)-nerolidol as major compound. Recently, we have shown that P. gaudichaudianum essential oil possesses strong cytotoxic effects in mammalian V79 cells. The aim of this study was to analyze the cytotoxicity and mutagenicity of P. gaudichaudianum essential oil and nerolidol using Saccharomyces cerevisiae as model study. Treatment of the XV185-14c and N123 strains with essential oil and nerolidol led to cytotoxicity but did not induce mutagenicity. Our results revealed an important role of base excision repair (BER) as the ntg1, ntg2, apn1 and apn2 mutants showed pronounced sensitivity to essential oil and nerolidol. In the absence of superoxide dismutase (in sod1Δ mutant strain) sensitivity to the essential oil and nerolidol increased indicating that this oil and nerolidol are generating reactive oxygen species (ROS). The ROS production was confirmed by DCF-DA probing assay in Sod-deficient strains. From this, we conclude that the observed cytotoxicity to P. gaudichaudianum essential oil and nerolidol is mainly related to ROS and DNA single strand breaks generated by the presence of oxidative lesions.

Plant growth and productivity are adversely affected by frequent exposure to various abiotic and biotic stress factors, such as heavy metals. Heavy metals are elements with a relatively high density and are toxic or poisonous. Different molecular mechanisms for heavy metal toxicity have been described, and, among these, the production of reactive oxygen species (ROS) deserves special attention. ROS are highly reactive atoms or molecules naturally produced in plants and predominantly formed in the electron transport chain of cellular respiration (chloroplasts) and in photoreactivation. Oxidative attack on DNA generates both altered bases and damaged sugar residues that undergo fragmentation and lead to strand breaks. DNA damage caused by exposure to ROS is one of the primary causes of DNA decay in most organisms. The irreversible DNA damage can interfere with plant development and affect crop productivity. To protect the cells, a complex network of proteins is activated for damage control and repair that includes both antioxidant enzymes removing ROS and DNA repair proteins. The DNA repair response includes different pathways, such as mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), nonhomologous end joining (NHEJ), and homologous recombination (HR). Recent advances in the study of DNA repair in higher plants show that they use similar mechanisms to those present in other eukaryotes to remove and/or tolerate oxidized bases and other oxidative DNA lesions. The aim of this chapter is to review the latest data regarding the induction of oxidative stress by heavy metals in higher plants, introducing some basic concepts and seeking a relationship with DNA damage repair mechanisms.

We have investigated the chemical composition of Piper gaudichaudianum essential oil, as well as its cytotoxic, mutagenic and genotoxic effects in V79 cells. The chemical analyses showed that the major compounds are (E)-nerolidol (22.4%), alpha-humulene (16.5%), (E)-caryophyllene (8.9%) and bicyclogermacrene (7.4%). Dose-dependent cytotoxic effects were observed in V79 cells treated with essential oil by using clonal survival, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide reduction assay (MTT) and trypan blue exclusion assay (TB), and a significant decrease in survival was observed at concentrations of 0.5 microg/mL and higher. The P. gaudichaudianum essential oil treatment caused DNA strand breaks in V79 cells at concentrations up to 2 microg/mL, as detected by the alkaline comet assay, but did not induce double-strand breaks, as verified by neutral comet assay. It induced a significant increase in the frequency of micronucleated cells at 4, 6 and 10 microg/mL. Moreover, P. gaudichaudianum essential oil significantly increased lipid peroxidation at doses of 0.5 microg/mL and higher, suggesting that the observed oxidant potential can be responsible, at least in part, for its cytotoxic and genotoxic effects.

Piperaceae family is original from tropical regions and it shows more than 700 species around the world. Piper gaudichaudianum Kunth is the specie more abundant in Brazil, occurring from Northeast to South Brazil. In this paper, it was investigated the influence of some experimental parameters on the pressurized liquid extraction (PLE) of P. gaudichaudianum Kunth leaves, using petroleum ether as extractor solvent. The optimization of the main variables involved in the PLE process (extraction temperature and time) has been done by response surface methodology (RSM) using, as responses, the extraction yield and the chromatographic profile (GC/MS) of the extracts. The optimized procedure employed 3 g of ground leaves, 10 min of extraction and one cycle of extraction at 85 °C. The major compounds present in the petroleum ether extracts were: palmitic acid, stearic acid and nerolidol. The results presented in this work show the possibility of using a fast and easy process to recover compounds from P. gaudichaudianum Kunth.