Effect of Non-Thermal Atmospheric Plasma on Food-Borne Bacterial Pathogens on Ready-to Eat Foods: Morphological and Physico-Chemical Changes Occurring on the Cellular Envelopes.

Currently, there is a need for new technological interventions to guarantee the microbiological safety of ready-to-eat (RTE) foods. Non-thermal atmospheric plasma (NTAP) has emerged as a promising strategy for inactivating microorganisms on thermo-sensitive foods, and the elucidation of its mechanisms of action will aid the rational optimization and industrial implementation of this technology for potential applications in the food industry. In this study, the effectiveness of NTAP for inactivating strains of Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes contaminating the surface of different sliced RTE foods ("chorizo", salami, bacon, smoked salmon, tofu and apple) was investigated. In addition, to further assess the bacterial inactivation mechanisms of NTAP, the morphological and physico-chemical damages in bacterial cells were analyzed. NTAP was effective for the surface decontamination of all products tested and, especially, of cut apple, where the microbial populations were reduced between 1.3 and 1.8 log units for the two Salmonella strains and E. coli O157: H7, respectively, after 15 min of exposure. In the rest of foods, no significant differences in the lethality obtained for the E. coli O157:H7 strain were observed, with inactivation rates of between 0.6 and 0.9 log cycles after a 15-min treatment. On the other hand, the strains from the rest of pathogenic microorganisms studied were extremely resistant on tofu, where barely 0.2-0.5 log units of inactivation were achieved after 15 min of plasma exposure. S. Enteritidis cells treated for 10 min exhibited noticeable morphological and structural changes, as observed by transmission electron microscopy, which were accompanied by a loss in membrane integrity, with an increased leakage of intracellular components and uptake of propidium iodide and marked changes in regions of their FTIR spectra indicating major alterations of the cell wall components. Overall, this indicates that loss of viability was likely caused for this microorganism by a significant damage in the cellular envelopes. However, the plasma-treated cells of L. monocytogenes did not show such obvious changes in morphology, and exhibited less marked effects on the integrity of their cytoplasmic membrane, what suggests that the death of this pathogenic microorganism upon NTAP exposure is more likely to occur as a consequence of damages in other cellular targets.

A Review on Non-thermal Atmospheric Plasma for Food Preservation: Mode of Action, Determinants of Effectiveness, and Applications.

Non-thermal Atmospheric Plasma (NTAP) is a cutting-edge technology which has gained much attention during the last decade in the food-processing sector as a promising technology for food preservation and maintenance of food safety, with minimal impact on the quality attributes of foods, thanks to its effectiveness in microbial inactivation, including of pathogens, spoilage fungi and bacterial spores, simple design, ease of use, cost-effective operation, short treatment times, lack of toxic effects, and significant reduction of water consumption. This review article provides a general overview of the principles of operation and applications of NTAP in the agri-food sector. In particular, the numerous studies carried out in the last decade aimed at deciphering the influence of different environmental factors and processing parameters on the microbial inactivation attained are discussed. In addition, this review also considers some important studies aimed at elucidating the complex mechanism of microbial inactivation by NTAP. Finally, other potential applications of NTAP in the agri-food sector, apart from food decontamination, are briefly described, and some limitations for the immediate industrial implementation of NTAP are discussed (e.g., impact on the nutritional and sensory quality of treated foods; knowledge on the plasma components and reactive species responsible for the antimicrobial activity; possible toxicity of some of the chemical species generated; scale-up by designing fit-for-purpose equipment).

Stress adaptation has a minor impact on the effectivity of Non-Thermal Atmospheric Plasma (NTAP) against Salmonella spp.

This study was aimed at studying the influence of gas composition (air and nitrogen) at different flow rates (5, 10 and 15Lm-1) and stress adaptation (growth under a wide range of temperatures [10-45°C] and acid conditions [up to pH4.5, using different organic acids] or short-term exposure to acid, cold or heat stress shocks) on the inactivation by Non-Thermal Atmospheric Plasma (NTAP) of S. Typhimurium CECT 443 and S. Enteritidis CECT 4300. Results obtained evidence that microbial inactivation was significantly higher when air was used for NTAP treatments. D-values obtained using air ranged from 0.86 to 2.43min and 0.90 to 1.69min for S. Typhimurium and S. Enteritidis, respectively, while those obtained using nitrogen ranged from 3.08 to 5.75min and 2.28 to 5.54min, respectively. Microbial inactivation also increased with increasing flow rates, although differences were not statistically significant in all cases. Growth temperature and pH or exposure to acid, cold or heat stress shocks had a minor impact on NTAP resistance. Indeed, D-values obtained under the different stress adaptation scenarios were not significantly different from those obtained for non-adapted control cultures (1.38±0.39 for S. Typhimurium and 1.23±0.36 for S. Enteritidis), with the exception of cells grown at 10°C, which were significantly more sensitive to NTAP, with D-values of 0.68±0.11 and 0.45±0.10min, respectively, for S. Typhimurium and S. Enteritidis. These findings suggest that adaptive responses triggered by exposure to acid, cold or heat stresses, already described in the past for these two Salmonella strains, do not provide protection against NTAP treatments, which allows us to conclude that NTAP may be a first-choice technology to be included into food processing schemes following a hurdles technology approach in combination with acidification, mild heating or refrigeration.

Effects of indirubin and isatin on cell viability, mutagenicity, genotoxicity and BAX/ERCC1 gene expression.

CONTEXT: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood. OBJECTIVE: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes. MATERIALS AND METHODS: HeLa and/or CHO-K1 cell lines were tested (3 or 24 h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72 h) tests after treatment with IRN (0.1 to 200 µM) or ISA (0.5 to 50 µM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24 h) by gavage (50, 100 and 150 mg/kg determined from the LD50 - 1 g/kg b.w.) and submitted to comet assay in vivo. RESULTS: IRN reduced the viability of CHO-K1 (24 h; 5 to 200 µM) and HeLa cells (10 to 200 µM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10 µM; HeLa: 5 and 10 µM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24 h) at all doses tested. IRN (100 and 150 mg/kg) also induced genotoxicity in vivo (4 h). CONCLUSION: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.

Influence of processing parameters and stress adaptation on the inactivation of Listeria monocytogenes by Non-Thermal Atmospheric Plasma (NTAP).

This study evaluated the effectiveness of Non-Thermal Atmospheric Plasma (NTAP) treatments against Listeria. Firstly, the impact of gas composition and flow rate on L. monocytogenes and L. innocua (used as a surrogate) inactivation by NTAP was monitored. Secondly, the influence of stress adaptation (growth under suboptimal conditions, using a wide range of temperatures and media acidified up to pH5.5 with citric, lactic, malic or hydrochloric acid, or short-term exposure to acid, cold or thermal shocks) on L. monocytogenes NTAP resistance was assessed. Survival curves obtained were concave upward. A mathematical model based on the Weibull distribution accurately described the inactivation kinetics. Both L. monocytogenes and L. innocua showed a higher sensitivity to plasma when the treatment was performed using air than when nitrogen was used. In fact, the use of nitrogen as working gas made the plasma treatment almost ineffective. The effect of gas flow rate on the effectiveness of the NTAP treatment depended on the type of gas used to generate plasma. Increases in flow rate from 5 to 10L/min caused an acceleration of bacterial inactivation when air was used, while an additional increase of gas flow from 10 to 15L/min had a minor impact on microbial inactivation. On the other hand, gas flow rate hardly affected NTAP treatment efficiency when nitrogen was used to generate plasma. L. monocytogenes growth under sub-optimal temperature or pH conditions or short-term exposure to acid, heat or cold stress conditions did not significantly modify its NTAP resistance. This suggests that temperature and pH stress adaptation does not induce a cross-protection response against NTAP treatments in L. monocytogenes, what makes NTAP an attractive technology for food decontamination within minimal processing strategies targeting this pathogenic microorganism.

Ulcer healing and mechanism(s) of action involved in the gastroprotective activity of fractions obtained from Syngonanthus arthrotrichus and Syngonanthus bisulcatus.

BACKGROUND: Syngonanthus arthrotrichus and Syngonanthus bisulcatus, currently known for Comanthera aciphylla (Bong.) L.R.Parra & Giul. and Comanthera bisulcata (Koern.) L.R. Parra & Giul, popularly known in Brazil as "sempre-vivas," are plants from the family Eriocaulaceae. They are found in the states of Minas Gerais and Bahia. The species are known to be rich in flavonoids to which their gastroprotective activity has been attributed. In this research, experimental protocols were performed to elucidate the associated mechanisms of action. METHODS: The activity was evaluated using induced gastric ulcer models (acetic acid and ethanol-induced gastric lesions in NEM or L-NAME pre-treated mice, and by ischemia/reperfusion). Antioxidant enzymes, serum somatostatin, and gastrin were also evaluated. RESULTS: In chronic gastric ulcers, a single daily oral dose of Sa-FRF or Sb-FRF (100 mg/kg body wt.) for 14 consecutive days accelerated ulcer healing to an extent similar to that seen with an equal dose of cimetidine. The pre-treatment of mice with NEM (N-ethylmaleimide) or L-NAME (N-nitro-L-arginine) abolished the protective activity of Sa-FRF, Sa-FDF, Sb-FDF and Sb-FRF or Sa-FRF and Sb-FRF, respectively, which indicates that antioxidant compounds and nitric oxide synthase activity are involved in the gastroprotective. Sa-FRF and Sb-FRF (100 mg/kg p.o) protected the gastric mucosa against ulceration that was induced by ischemia/reperfusion (72 and 76 %, respectively). It also decreased lipid peroxidation and restored total thiols in the gastric wall of mice that had been treated with ethanol. When administered to rats submitted to ethanol-induced gastric lesions, Sa-FRF and Sb-FRF (100 mg/kg, p.o.) increased the somatostatin serum levels, while the gastrin serum levels were proportionally decreased. CONCLUSIONS: The results indicate significant healing effects and gastroprotective activity for the Sa-FRF and Sb-FRF, which probably involves the participation of SH groups, nitric oxide (NO), the antioxidant system, somatostatin, and gastrin. All are integral parts of the gastrointestinal mucosa's cytoprotective mechanisms against aggressive factors.

In vitro assessment of the cytotoxic, apoptotic, and mutagenic potentials of isatin.

Isatin (1H-indole-2,3-dione) is a chemical found in various medicinal plant species and responsible for a broad spectrum of pharmacological and biological properties that may be beneficial to human health, as an anticonvulsant, antibacterial, antifungal, antiviral, and anticancer agent. The aim of the present study was to determine in vitro the cytotoxic, mutagenic, and apoptotic effects of isatin on CHO-K1 and HeLa cells using the MTT viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), micronucleus (MN) test, apoptosis index, and nuclear division index (NDI). The 5 isatin concentrations evaluated in the mutagenicity and apoptosis tests were 0.5, 1, 5, 10, and 50 µM, selected through a preliminary MTT assay. Positive (doxorubicin, DXR) and negative (phosphate buffered saline, PBS) control groups were also included in the analysis. Isatin did not exert a mutagenic effect on CHO-K1 after 3 and 24 h of treatment or on HeLa cells after 24 h. However, 10 and 50 µM concentrations inhibited cell proliferation and promoted apoptosis in both CHO-K1 and HeLa cells. Data indicate that the cytotoxic, apoptotic, and antiproliferative effects of isatin were concentration independent and cell line independent.

Healing, antioxidant and cytoprotective properties of Indigofera truxillensis in different models of gastric ulcer in rats.

The present study evaluated the antiulcerogenic activity and mechanisms of the aqueous (AqF 100 mg/kg) and ethyl acetate (AcF 50 mg/kg) fractions from Indigofera truxillensis leaves. This dose was selected to assess its activity on ulcer healing and its action on gastric acid and mucus secretion, prostaglandin production and antioxidant enzyme activity (superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd)). Gastric ulcer was induced by absolute ethanol. Antisecretory action, mucus and prostaglandin production, healing and antioxidant enzyme activities were evaluated for both fractions. AqF and AcF significantly inhibited the gastric mucosal damage caused by ethanol. This effect was statistically significant at 100 and 50 mg/kg compared with the vehicle. Neither fraction interfered with gastric secretion. AcF increased the PGE(2) production, and both fractions increased mucus production. l-NAME did not alter the gastroprotection exerted by the fractions, but N-ethylmaleimide attenuated only AcF. In the ischemia/reperfusion model both fractions inhibited the mucosal damage. AcF increased SOD, GSH-Px and GSH-Rd activity, but AqF increased only SOD and GSH-Px. In the acetic acid-induced ulcer model AcF only accelerated ulcer healing. These results showed that Indigofera truxillensis acted as a gastroprotective agent, stimulating protective factors and antioxidants enzymes.

Immunostimulatory and cytotoxic activities of Indigofera suffruticosa (Fabaceae).

One of the major disadvantages of the current cancer therapy is the suppression of the immune system. Brazilian flora is considered one of the most diverse in the world and many plants were found to contain active constituents that can be valuable sources of new drugs. The plant Indigofera suffruticosa was studied to determine its potential to stimulate the immune system and also to be effective against tumour cells. We investigated the effects of the alkaloidal fraction and the pure alkaloid indigo obtained from I. suffruticosa on macrophage activation by measuring nitric oxide (NO) and tumour necrosis factor-α (TNF-α) production. Cytotoxic activity was also evaluated against the two tumour murine cells lines, LM2 (breast adenocarcinoma) and LP07 (lung adenocarcinoma). The alkaloidal fraction induced a high NO production and a moderated TNF-α release. The pure indigo demonstrated an elevated NO and TNF-α production. The fraction and the pure compound also exhibited cytotoxic activity against both adenocarcinoma cell lines and indigo showed the strongest cytotoxic activity with IC50 value of 0.89 µg mL⁻¹ against LM2 and 1.44 µg mL⁻¹ against LP07. Our results presented the immunostimulatory and cytotoxic activity of I. suffruticosa, enhancing macrophage function and therefore contributing to the host defence against tumours.

Indigofera suffruticosa Mill as new source of healing agent: involvement of prostaglandin and mucus and heat shock proteins.

ETHNOPHARMACOLOGICAL RELEVANCE: Indigofera suffruticosa is specie typical of the "Cerrado" or Brazilian savannah; it is a member of the Fabaceae family - in folkmedicine is used for gastric disorders, infection and inflammation. AIM OF THE STUDY: Ethyl acetate fraction (AcF) and aqueous fraction (AqF) of the methanolic extract of I. suffruticosa leaves were evaluated against acute gastric ulcer. The AcF fraction was selected to assess its activity in ulcer healing and its gastroprotective effects via mucus and gastric secretion. MATERIALS AND METHODS: The gastroprotective action of AcF and AqF fractions were evaluated in a rodent experimental model. The action mechanisms, involvements of the antisecretory action, mucus and prostaglandin production, toxicological and healing activity of the AcF (100mg/kg, p.o.) were evaluated. We also used histological analysis (HE and PAS) and immunohistochemical (PCNA and HSP-70) assays to evaluate the effects of I. suffruticosa. RESULTS: AcF significantly inhibited the gastric mucosal damage caused by ethanol. This effect was statistically significant in 100mg/kg group compared vehicle. AcF did not interfered with gastric secretion, significantly increased the PGE(2) and mucus production (validated in PAS technique). The gastroprotection was attenuated by pretreatment with N-ethylmaleimide, but not L-NAME. In acid-acetic-induced ulcer model AcF accelerated ulcer healing. Immunohistochemistry analysis showed induction of proliferating cell (PCNA) and heat shock protein (HSP 70). CONCLUSIONS: These results showed that AcF acted as gastroprotective agent stimulating prostaglandin, mucus and HSP70.

Mutagenic Activity of Indigofera truxillensis and I. suffruticosa Aerial Parts.

Indigofera truxillensis and I. suffruticosa, are used as a source of indigo dye and to treat several diseases. The mutagenic activity of the methanolic extracts from aerial parts, glycerolipid, flavonoid and alkaloid fractions of the extract were evaluated by means of Salmonella/microsome assays using TA100, TA98, TA102 and TA97a strains. The methanolic extract of I. truxillensis showed mutagenic activity in the TA98 strain without S9 while glycerolipid fraction was devoid of activity. The flavonoid and alkaloid fractions of both plants showed mutagenicity. Chemical analysis of flavonoid fractions of I. truxillensis and I. suffruticosa resulted in the identification of kaempferol, quercetin and their derivatives. The alkaloid fraction of both the species contained indigo and indirubin and indigo was found mainly responsible for the mutagenic activity.

Effects of the Ethyl Acetate Fraction of Alchornea triplinervia on Healing Gastric Ulcer in Rats.

Alchornea triplinervia (Spreng.) Muell. Arg (Euphorbiaceae) is a medicinal plant commonly used by people living in the Cerrado region of Brazil to treat gastrointestinal ulcers. We previously described the gastroprotective action of methanolic extract (ME) of Alchornea triplinervia and the ethyl acetate fraction (EAF) in increasing of prostaglandin E2 (PGE2) gastric levels in the mucosa. In this work we evaluated the effect of EAF in promoting the healing process in rats with acetic acid-induced gastric ulcers. In addition, toxicity was investigated during treatment with EAF. After 14 days of treatment with EAF, the potent stimulator of gastric cell proliferation contributed to the acceleration of gastric ulcer healing. Upon immunohistochemical analysis, we observed a pronounced expression of COX-2, mainly in the submucosal layer. The 14-day EAF treatment also significantly increased the number of neutrophils in the gastric mucosa regeneration area. The EAF induced angiogenesis on gastric mucosa, observed as an increase of the number of blood vessels supplying the stomach in rats treated with EAF. Oral administration for 14 days of the ethyl acetate fraction from Alchornea triplinervia accelerated the healing of gastric ulcers in rats by promoting epithelial cell proliferation, increasing the number of neutrophils and stimulation of mucus production. This fraction, which contained mainly phenolic compounds, contributed to gastric mucosa healing.

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo.

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.

Phenolic compounds in leaves of Alchornea triplinervia: anatomical localization, mutagenicity, and antibacterial activity.

Phenolic compounds are produced by secretory idioblasts and hypodermis, and by specialized cells of the epidermis and chlorenchyma of leaves of Alchornea triplinervia. Phytochemical investigation of these leaves led to the isolation of the known substances quercetin, quercetin-7-O-beta-D-glucopyranoside, quercetin-3-O-beta-D-glucopyranoside, quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, brevifolin carboxylic acid, gallic acid, and methyl gallate from the methanolic extract, and stigmasterol, campesterol, sitosterol, lupeol, friedelan-3-ol, and friedelan-3-one from the chloroform extract. In studies of antibacterial activity and mutagenicity, the methanolic extract showed promising activity against Staphylococcus aureus (MIC = 62.5 microg/mL) and was slightly mutagenic in vitro and in vivo at the highest concentrations tested (1335 mg/kg b.w.).

Anti-inflammatory activity of Alchornea triplinervia ethyl acetate fraction: inhibition of H2O2, NO and TNF-α.

CONTEXT: Alchornea triplinervia (Spreng.) Müll. Arg. (Euphorbiaceae) is a tree widespread in many Brazilian states. This plant naturally occurs in different ecosystems including tropical Atlantic forest, Amazon rain forest, moist tropical mixed forest, savanna forest, among others. Local populations traditionally use it in tea form to treat gastric disturbances. OBJECTIVE: The objective of this research was to evaluate the plant A. triplinervia as a potential inhibitor of some macrophage functions involved in the inflammatory process. MATERIALS AND METHODS: The effects of Alchornea triplinervia ethyl acetate fraction (AtF) on hydrogen peroxide (H2O2), nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in peritoneal macrophages were investigated using phenol red, Griess reagent and a sandwich immunoassay, respectively. RESULTS: AtF chromatographic analyses indicate the presence of flavonoids as majority compounds. The fraction also showed an intense inhibition of H2O2 and NO production. The inhibitory effects of the fraction in H2O2 and NO production ranged from 72.25 ± 4.68 to 69.64 ± 4.21 and from 47.8 ± 8.96 to 76.77 ± 8.11%, respectively in the two tested concentrations, 15.62 and 62.5 µg/mL. TNF-α production was partially inhibited in the tested concentrations and the inhibitory rate was around 18%. DISCUSSION AND CONCLUSION: It is supposed that the elevated biological potential of A. triplinervia is related to the presence of phenolic compounds in the plant leaves. According to the results observed in this study, it is suggested that AtF presents anti-inflammatory activity, supporting the traditional use of A. triplinervia in Brazilian folk medicine.

Antimycobacterial activity of Indigofera suffruticosa with activation potential of the innate immune system.

Mycobacterium tuberculosis is responsible for over 8 million cases of tuberculosis (TB) annually. Natural products may play important roles in the chemotherapy of TB. The antimycobacterial activity and the innate immune response of methanol (METH) and dichloromethane (DCM) extracts of Indigofera suffruticosa Miller (Fabaceae) were evaluated. We observed that the minimum inhibitory concentrations (MICs) for METH and DCM extracts were 125 and 1000 microg/mL, respectively. However, they were able to induce the innate immune response through the production of high levels of NO and TNF-alpha (p < 0.001) by peritoneal exudate cells (PECs). These results suggest that I. suffruticosa extracts may have an important immunological role in the control of TB once macrophage activity is induced by them.

Mutagenicity of two species of the genus Alchornea measured by Salmonella microsome assay and micronucleus test / Mutagenicidade de duas espécies do gênero Alchornea avaliadas através de ensaios com Salmonella microssomo e teste do micronúcleo

Some species of the plant genus Alchornea (family Euphorbiaceae) are widely used in popular medicine, mainly in South America and in Africa. Several kinds of biological activity have been seen in the species: antioxidant, antifungal, anti-inflammatory, antibacterial, cytotoxic against tumor cell lines and inhibitory to the replication of HIV-1 and HIV-2. In Brazil, the species Alchornea castaneaefolia Willd. A. Juss. and Alchornea glandulosa Poepp. & Endl. are used by the local population to treat rheumatism, arthritis and muscular pains. In view of the popular use of these plants as medicines and the potential risks from their consumption, we assessed the mutagenic potential of chloroform and methanol extracts of the leaves of these plant species, employing the in vivo micronucleus test and the Ames assay. The data obtained showed that the chloroform extracts were not mutagenic. The methanol extract of A. castaneaefolia was mutagenic to strain TA98 of Salmonella typhimurium and the methanol extract of A. glandulosa to strains TA98 and TA97a. The methanol extracts of both species of Alchornea were mutagenic in vivo at the largest dose employed. The probable mutagenic agents involved were the aglycone quercetin and amentoflavone, present in both species.

Algumas espécies de plantas do gênero Alchornea (Euphorbiaceae) são conhecidas por apresentarem as atividades biológicas: antioxidante, antifúngica, antiinflamatória, antibacteriana, citotóxica para células tumorais e inibidoras da replicação dos vírus HIV-1 e HIV-2. São também amplamente usadas na medicina popular na America do Sul e África. No Brasil, Alchornea castaneaefolia Willd. A. Juss. e Alchornea glandulosa Poepp. & Endl. são usadas para tratamento do reumatismo, artrite e dores musculares. Devido ao uso medicinal dessas plantas e o potencial risco do seu consumo indiscriminado, no presente trabalho foi avaliada a atividade mutagênica dos extratos metanólico e clorofórmico das folhas, empregando o teste do micronúcleo in vivo e o teste de Ames. Os resultados mostraram que o extrato clorofórmico não apresentou mutagenicidade, porém, o extrato metanólico de A. castaneaefolia foi mutagênico para a linhagem TA98 de Salmonella typhimurium e o extrato metanólico de A. glandulosa para as linhagens TA98 e TA97a. O extrato metanólico de ambas as espécies também apresentaram mutagenicidade positiva nos ensaios in vivo na maior concentração usada. Os prováveis agentes mutagênicos envolvidos foram a quercetina aglicona e amentoflavona presentes em ambas as espécies.

Antioxidant activity of indigo and its preventive effect against ethanol-induced DNA damage in rat gastric mucosa.

Ethanol-induced oxidative damage is commonly associated with the generation of reactive oxygen molecules, leading to oxidative stress. Considering that antioxidant activity is an important mechanism of action involved in cytoprotection, the aim of this work was to evaluate the antioxidant properties of the alkaloid indigo (1) (2 mg/kg, P. O.), obtained from the leaves of Indigofera truxillensis Kunth (Fabaceae), on rat gastric mucosa submitted to ethanol-induced (100%, 1 mL, P. O.) gastric ulcer. Enzymatic assays and DNA fragmentation analysis were performed. When ethanol was administered to the control group, the sulfhydryl content (SH) and the glutathione peroxidase (GPx) activity decreased by 41% and 50%, respectively; in contrast, superoxide dismutase (SOD) and glutathione reductase (GR) activities increased by 56% and 67%, respectively. Additionally, myeloperoxidase (MPO) activity, a marker for free radical generation caused by polymorphonuclear neutrophil (PMN) tissue infiltration, also increased 4.5-fold after ethanol treatment. Rat gastric mucosa exposed to ethanol showed DNA fragmentation. Indigo alkaloid pretreatment protected rats from ethanol-induced gastric lesions. This effect was determined by the ulcerative lesion area (ULA), indicating an inhibition of around 80% at 2 mg/kg. This alkaloid also diminished GPx activity, which was higher than that observed with ethanol alone. However, this effect was counterbalanced by increased GR activity. Indigo was unable to restore alterations in SOD activity promoted by ethanol. After indigo pretreatment, SH levels and MPO activity remained normal and gastric mucosa DNA damage caused by ethanol was also partially prevented by indigo. These results suggest that the gastroprotective mechanisms of indigo include non-enzymatic antioxidant effects and the inhibition of PMN infiltration which, in combination, partially protect the gastric mucosa against ethanol-induced DNA damage.

Constituents and antiulcer effect of Alchornea glandulosa: activation of cell proliferation in gastric mucosa during the healing process.

Alchornea glandulosa (Euphorbiaceae) is a plant used in folk medicine as an antiulcer agent. Rats pretreated with methanolic extract obtained from the leaves of A. glandulosa (AG) showed a dose-dependent effect and significant reduction of gastric ulcers induced by absolute ethanol at the doses of 500 (57%) and 1000 mg/kg (85%) in relation to the control group. Pretreatment of mice with AG (500, 1000 mg/kg, p.o.) showed dose-dependent activity and significantly decreased the severity of lesions caused by HCl/ethanol and by non steroidal anti inflammatory drug-induced gastric lesions. Pretreatment with AG also induced antisecretory action via local and systemic routes and a significant decrease in the total gastric acid content. The gastroprotective effects of AG involved the participation of nitric oxide and increased levels of endogenous sulfhydryl compounds, which are defensive mechanisms of the gastrointestinal mucosa against aggressive factors. The ability of AG to heal gastric ulcers was evaluated after 14 consecutive days of treatment. The results showed that single oral administrations of AG (250 mg/kg/once daily) potently stimulates gastric epithelial cell proliferation that contributes to the accelerated healing of gastric ulcers induced by acetic acid. In addition, no subacute toxicity (body weight gain, vital organs, and serum biochemical parameters) was observed during treatment with AG. Phytochemical investigation of AG led to the isolation of myricetin-3-O-alpha-L-rhamnopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, quercetin-3-O-beta-D-galactopyranoside, quercetin, amentoflavone, methyl gallate, gallic acid, and pterogynidine. We also established the phytochemical profile of AG with the quantification of total phenolic compounds. These compounds may contribute to the observed antiulcerogenic effects of AG.

Antiulcerogenic activity of Alchornea castaneaefolia: effects on somatostatin, gastrin and prostaglandin.

The hydroethanolic extract of the leaves (HEL) and bark (HEB) obtained from Alchornea castaneaefolia (Euphorbiaceae) were investigated for their ability to prevent ulceration of the gastric mucosa in animal models. HEL (500 and 1000 mg/kg) and HEB (1000 mg/kg) significantly reduced the gastric injuries induced by the combination of HCl/ethanol and lowered the severity of gastric damage formation induced by indomethacin/bethanechol in mice. Further investigation showed that HEL also inhibited formation of ulcers in mice submitted to stress and pylorus ligature, but HEL did not modify gastric juice parameters in Shay mice. HEL was also effective in promoting the healing process in chronic gastric ulcer induced by acetic acid in rats. An enriched flavonoidic fraction (EFF at dose of 100mg/kg) obtained from HEL reduced gastric lesions induced by HCl/ethanol and indomethacin/bethanechol in mice. Although EFF did not modify the amount of free mucus production by gastric mucosa, it was able to increase prostaglandin production. When administered to rats submitted to ethanol-induced gastric lesions, EFF increased the somatostatin serum levels, while the gastrin serum levels were proportionally decreased. Phytochemical investigation on HEL and EFF led to the isolation of flavonoids glycosides as the main compounds, thus suggesting that these substances may be involved in the observed antiulcer activity.