Evaluation of acute toxicity, 28-day repeated dose toxicity, and genotoxicity of Moringa oleifera leaves infusion and powder.

ETHNOPHARMACOLOGICAL RELEVANCE: Moringa oleifera Lam. leaves infusion and powder are widely used by population due the nutritional and medicinal potentials, however data regarding safety of use are still inconclusive, leading to prohibition of this plant in some countries. AIM OF THE STUDY: The present work investigated the nutritional and phytochemical composition, acute and 28-day repeated dose toxicity, and genotoxicity of M. oleifera leaves infusion and powder. MATERIALS AND METHODS: For nutritional characterization of leaf powder, it was determined: humidity; mineral residue (ash); total lipid, protein, carbohydrate, and crude fiber contents; and total caloric value. Phytochemical composition was determined by high performance liquid chromatography (HPLC). The acute toxicity assay used Swiss female albino mice and oral administration in a single dose at 2000 and 5000 mg/kg of infusion or powder. The 28-day repeated dose toxicity assay employed female and male mice, with oral administration of infusion or powder at the doses 250, 500 and 1000 mg/kg. The animals were evaluated for body weight, water and feed consumption, biochemical and hematological parameters, and histology of the liver, spleen, and kidneys. In vivo genotoxicity and mutagenicity (2000 mg/kg) were evaluated by the comet assay and the micronucleus test, respectively. RESULTS: Nutritional characterization confirmed that M. oleifera leaves are rich in proteins, carbohydrates, lipids, minerals, and fiber. HPLC indicated the presence of flavonoids and cinnamic derivatives as major polyphenols. Acute toxicity did not reveal alterations in weight gain and water and feed consumptions and no change in biochemical, hematological, and histological parameters. Behavior alterations was observed in the first 2 h after administration at 5000 mg/kg in both treatments. Infusion did not present toxicity when administered for 28 days. Conversely, the powder at 500 and 1000 mg/kg promoted liver and kidney damages observed through biochemical parameters and histopathology. Genotoxicity and mutagenicity were not detected at 2000 mg/kg. CONCLUSIONS: The present study reveals that M. oleifera leaves are an important source of polyphenols and nutrients. Indiscriminate use of both infusion and crude leaf powder above 2000 mg/kg and powder at 500 and 1000 mg/kg are not recommended. Chronic toxicological studies and establishment of preparation protocols are suggested aiming to guarantee the safety in the use of M. oleifera leaves as nutraceutical by population.

Neurolocomotor Behavior and Oxidative Stress Markers of Thiazole and Thiazolidinedione Derivatives against Nauphoeta cinerea.

Thiazolidine compounds NJ20 {(E)-2-(2-(5-bromo-2-methoxybenzylidene)hydrazinyl)-4-(4-nitrophenyl)thiazole} and NW05 [(2-(benzo (d) (1,3) dioxol-4-ylmethylene)-N-(4-bromophenyl)-thiosemicarbazone] potentiated the effect of norfloxacin in resistant bacteria; however, there are no reports on their effects on Nauphoeta cinerea in the literature. The objective of this work was to evaluate the behavioral effects and oxidative markers of NW05 and NJ20 in lobster cockroach N. cinerea. To evaluate the behavioral study, a video tracking software was used to evaluate the locomotor points and the exploratory profile of cockroaches in the horizontal and vertical regions of a new environment. The total concentration of thiol and reduced glutathione (GSH), substances reactive to thiobarbituric acid (TBARS), free iron (II) content and mitochondrial viability were determined. The antioxidant potential was evaluated by the DPPH method. Both substances induced changes in the behavior of cockroaches, showing a significant reduction in the total distance covered and in the speed. In the cell viability test (MTT), there was a significant reduction for NJ20 (1 mM). NJ20 caused a significant increase in total levels of thiol and non-protein thiol (NPSH), although it also slightly increased the content of malondialdehyde (MDA). Both compounds (NW05 and NJ20) caused a significant reduction in the content of free iron at a concentration of 10 mM. In conclusion, the compound NJ20 caused moderate neurotoxicity (1 mM), but had good antioxidant action, while NW05 did not show toxicity or significant antioxidant activity in the model organism tested. It is desirable to carry out complementary tests related to the antioxidant prospection of these same compounds, evaluating them at different concentrations.

Antinociceptive Effects of Aza-Bicyclic Isoxazoline-Acylhydrazone Derivatives in Different Models of Nociception in Mice.

BACKGROUND: In a study recently published by our research group, the isoxazoline-acylhydrazone derivatives R-99 and R-123 presented promising antinociceptive activity. However, the mechanism of action of this compound is still unknown. OBJECTIVE: This study aimed to assess the mechanisms involved in the antinociceptive activity of these compounds in chemical models of pain. METHODS: Animals were orally pretreated and evaluated in the acetic acid-, formalin-, capsaicin-, carrageenan- and Complete Freund's Adjuvant (CFA)-induced pain models in mice. The effects of the compounds after pretreatment with naloxone, prazosin, yohimbine, atropine, L-arginine, or glibenclamide were studied, using the acetic acid-induced writhing test to verify the possible involvement of opioid, α1-adrenergic, α2-adrenergic or cholinergic receptors, and nitric oxide or potassium channels pathways, respectively. RESULTS: R-99 and R-123 compounds showed significant antinociceptive activity on pain models induced by acetic acid, formalin, and capsaicin. Both compounds decreased the mechanical hyperalgesia induced by carrageenan or CFA in mice. The antinociceptive effects of R-99 and R-123 on the acetic acid-induced writhing test were significantly attenuated by pretreatment with naloxone, yohimbine or atropine. R-99 also showed an attenuated response after pretreatment with atropine and glibenclamide. However, on the pretreatment with prazosin, there was no change in the animals' response to both compounds. CONCLUSION: R-99 and R-123 showed antinociceptive effects related to mechanisms that involve, at least in part, interaction with the opioid and adrenergic systems and TRPV1 pathways. The compound R-99 also interacts with the cholinergic pathways and potassium channels.

Antioxidant, Antimicrobial, and Anticancer Effects of Anacardium Plants: An Ethnopharmacological Perspective.

Anacardium plants have received increasing recognition due to its nutritional and biological properties. A number of secondary metabolites are present in its leaves, fruits, and other parts of the plant. Among the diverse Anacardium plants' bioactive effects, their antioxidant, antimicrobial, and anticancer activities comprise those that have gained more attention. Thus, the present article aims to review the Anacardium plants' biological effects. A special emphasis is also given to their pharmacological and clinical efficacy, which may trigger further studies on their therapeutic properties with clinical trials.

Acute and repeated dose 28-day oral toxicity of Chrysobalanusicaco L. leaf aqueous extract.

Chrysobalanus icaco L. is a native plant of Brazil used as a food source and traditionally for the treatment of various diseases. The aim of study was performed the phytochemical analysis by UPLC-DAD-ESI-QTOF-MS/MS, and evaluated acute and repeated dose oral toxicities of the C. icaco L. leaf aqueous extract (AECi). The acute toxicity study was performed using a dose of AECi 2000 mg/kg, while the repeated dose toxicity study, the AECi was administered daily at doses of 100, 200 and 400 mg/kg, for 28 days. Behavior and mortality of animals were observed during the test period and body weight, as well water and eating consumption. Hematological, biochemical parameters and histopathological examinations were carried out. Phytochemical analysis of the AECi revealed the presence of flavonoids and tannins. Oral single dose of 2000 mg/kg of AECi resulted in no mortalities or abnormal clinical signs. Studies of repeated dose toxicity promoted a reduction in the body weight of treated animals and an increase of hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in both, males and females. Histopathological analyzes showed alterations in the livers of animals treated with AECi. Thus, this study recommends the population take care when using this species, especially during prolonged periods.

Gastroprotective effect of ethyl acetate extract from Avicennia schaueriana Stapf & Leechman and underlying mechanisms.

OBJECTIVE: Avicennia schaueriana Stapf is an endemic mangrove species widely used by traditional Brazilian communities as a folk remedy for the treatment of rheumatism, ulcers, and skin wounds. The aim of the present study was to evaluate the gastroprotective potential of the ethyl acetate extract from the leaves of A. schaueriana (As-AcOEt). METHODS: Ultra-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF-MS/MS) was performed to identify chemical constituents of the ethyl acetate extract from the leaves ofA. schaueriana. Total phenols, flavonoids and tannins were determined and antioxidant activity was evaluated using the DPPH and ABTS methods. The acute toxicity of As-AcOEt and gastroprotective activity on HCl/ethanol-induced gastric ulcers were assessed and mechanisms of action involving the role of nitric oxide, sulfhydryl compounds, and prostaglandins were investigated. RESULTS: Terpenes, flavonoids and tannins were detected in the extract. As-AcOEt exhibited antioxidant activity, with an EC50 of 42.2 ± 4.4 µg/mL (DPPH) and 73.2% inhibition of ABTS radicals. UPLC-DAD-QTOF-MS/MS analysis identified gallic acid, gallic acid derivative, ellagic acid, myricetin pentoside, myricetin deoxyhexose, quercetin pentoside, quercetin deoxyhexose, and other compounds. Gallic acid was isolated in this species for the first time. During the acute toxicity test, no deaths or changes occurred in the variables evaluated. In the ethanol-induced ulcer model, As-AcOEt reduced the ulcerative lesion index, with 50, 100 and 200 mg/kg achieving 83.8, 88.5 and 86.9% inhibition, respectively. MPO levels decreased and the gastric mucosa of the animals treated with the extract was preserved. Pre-treatment with N-omega-nitro-l-arginine methyl ester (L-NAME; NO blocker) or carbenoxolone (CBXN; NP-SH blocker) reversed the gastroprotective effect of As-AcOEt, but this effect was not reversed with the previous administration of indomethacin. CONCLUSION: The present findings reveal that the extract from the leaves ofA. schaueriana has gastroprotective effects, suggesting the involvement of nitric oxide and nonprotein sulfhydryl compounds, but not prostaglandin. Therefore, the use of A. schaueriana in Brazilian folk medicine for the treatment of gastric disorders has a scientific basis.

Anti-inflammatory and antinociceptive activities of the leaf methanol extract of Miconia minutiflora (Bonpl.) DC. and characterization of compounds by UPLC-DAD-QTOF-MS/MS.

Some species of the genus Miconia are used in Brazilian folk medicine as analgesic and anti-inflammatory; however, several species of this genus are still poorly studied. Therefore, the aims of this study were to investigate the phytochemistry characterization by UPLC-DAD-QTOF-MS/MS, acute toxicity, anti-inflammatory and antinociceptive properties of Miconia minutiflora (Bonpl.) DC. The methanol extract of M. minutiflora (Mm-MeOH) was subjected to ultra-high-performance liquid chromatography (UPLC-DAD-QTOF-MS/MS) for the identification of the main phytocompounds. The anti-inflammatory properties of the extracts were studied using several inflammation models induced by carrageenan and acetic acid-induced vascular permeability. Antinociceptive effects of Mm-MeOH were assessed in nociception induced by intraperitoneal acetic acid or subplantar formalin injection. The role of α-adrenergic, cholinergic, and opioid receptors in modulating the extract's antinociceptive activity was determined. Analyses by UPLC-DAD-QTOF-MS/MS revealed the presence of ellagic acid, gallotannin, and terpenes in the methanol extract. Mm-MeOH (100 mg/kg) reduced carrageenan-induced paw edema and vascular permeability and inhibited leukocyte migration toward the air pouch and pleural cavity. Furthermore, Mm-MeOH decreased tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels. Administration of Mm-MeOH reduced the number of writhes by 58.9% and increased the pain threshold in the formalin test. The anti-inflammatory action mechanism of Mm-MeOH is associated with inhibition of proinflammatory cytokines TNF-α and IL-1ß, whereas the antinociceptive actions involve peripheral and central mechanisms with participation of α2-adrenergic receptors. These effects may be attributed to the presence of polyphenolics in the extract.

Antiulcer Activity and Potential Mechanism of Action of the Leaves of Spondias mombin L.

Spondias mombin L. is used in folk medicine for the treatment of inflammation and gastrointestinal diseases. Our study investigated the antiulcer activity of S. mombin ethanolic extract (SmEE) and its majority compounds gallic acid (GA) and ellagic acid (EA). Phytochemical characterization was performed by HPLC. The SmEE was screened for in vitro antioxidant activities using phosphomolybdenum, ABTS, DPPH, and FRAP assays. The antiulcer activity of SmEE, GA, EA, or GA + EA was evaluated by gastric lesion models induced by absolute ethanol and indomethacin. Following this, it is capable of stimulating mucus production, antisecretory capacity, and the influence of -SH groups and NO in the effect of SmEE. Its healing activity was demonstrated by acetic acid-induced chronic ulcer model. Anti-Helicobacter pylori activity was assessed by determining the MIC of the SmEE (64-1024 µg/mL). The HPLC results identified the presence of gallic acid and ellagic acid in SmEE. The extract showed antioxidant activity in vitro. SmEE (50, 100, and 200 mg/kg) reduced the area of ulcerative lesions induced by ethanol in 23.8, 90.3, and 90.2%, respectively. In NSAID model, the SmEE induced protection of 36.8, 49.4, and 49.9%, respectively. GA (10 mg/kg) or EA (7 mg/kg) or the association of GA + EA (10 + 7 mg/kg) inhibited the ethanol-induced lesions in 71.8, 70.9, and 94.9%, respectively, indicating synergistic action. SmEE (100 mg/kg) decreased acid secretion and H+ concentration in the gastric contents, increased levels of mucus, and showed to be dependent of -SH groups and NO on the protection of the gastric mucosa. In chronic ulcer model, SmEE reduced the gastric area lesion. SmEE showed anti-H. pylori activity. In conclusion, our study showed that SmEE has antiulcerogenic activity. GA and EA are isolated gastric protectors and, when associated, acted synergistically to protect the gastric mucosa.

Anti-inflammatory and anti-edematogenic action of the Croton campestris A. St.-Hil (Euphorbiaceae) essential oil and the compound ß-caryophyllene in in vivo models.

BACKGROUND: Inflammation makes up a set of vascularized tissue reactions acting in the defense of the body against harmful stimuli. Natural products are a lower cost alternative with better benefit, often used in popular medicine in the treatment of inflammatory processes. Several species from the genus Croton have scientifically proven anti-inflammatory action. PURPOSE: This study aims to analyze the chemical composition of the Croton campestris A. St.-Hil essential oil (EOCC), derived from fresh leaves, as well as to evaluate the anti-inflammatory potential and the possible mechanisms of action of the EOCC and its constituent ß-caryophyllene. METHODS: The assays were performed in in vivo models of acute and chronic inflammation. Initially, the chemical composition of the EOCC was determined and its oral toxicity was evaluated, followed by the evaluation of its topical antiedematogenic effect through acute and chronic ear edema induced by Croton oil. For the systemic verification of an anti-inflammatory action, the abdominal contortions, formalin test, paw edema induced by carrageenan, dextran, histamine and arachidonic acid models, as well as a peritonitis test, vascular permeability and granuloma assays were performed. RESULTS: The evaluation of the essential oil chemical composition revealed the presence of ß-caryophyllene (15.91%), 1,8-cineol (16.98%) and germacrene-D (14.51%) as its main constituents. The EOCC had no relevant clinical toxicity on oral administration, with an LD50 of more than 5000 mg/kg. The tested substances showed anti-inflammatory action in the abdominal contortions, paw edema induced by carrageenan, dextran, histamine and arachidonic acid models, the formalin test, peritonitis test and vascular permeability; however, ß-caryophyllene had no significant effect on the granuloma assay. This suggests as a hypothesis that both substances tested showed significant influence on the arachidonic acid and histamine pathway reducing edema in these models. CONCLUSION: The tested substances have a clinically safe profile, additionally the EOCC and ß-caryophyllene presented relevant anti-inflammatory activity. This study supports the hypothesis that ß-caryophyllene, in association with other constituents present in the EOCC such as 1,8-cineole, contributed to the anti-inflammatory effect observed, in addition to suggesting that one of the mechanisms of action probably involves the inhibition of cytokines with the involvement of the arachidonic acid and histamine pathways.

Spondias purpurea L. (Anacardiaceae): Antioxidant and Antiulcer Activities of the Leaf Hexane Extract.

Spondias purpurea is used in folk medicine to treat diarrhea and diuresis. The objective of this study was to evaluate the phytochemical profile and antioxidant and antiulcer activities of the hexane extract of the leaves of S. purpurea (SpHE). Phytochemical profile was evaluated via thin layer chromatography (TLC) and HPLC. SpHE was screened for antioxidant activities using DPPH, ABTS, FRAP, and phosphomolybdenum assays. To determine its antiulcer properties, animals were pretreated with injured control, lansoprazole, ranitidine, carbenoxolone, or SpHE (12.5, 25, and 50 mg/kg) and were screened; acute ulcers were induced by HCl/ethanol, absolute ethanol, and nonsteroidal anti-inflammatory drug (NSAID). TLC revealed the presence of flavonoids, whereas HPLC analysis showed the presence of caffeic acid and epigallocatechin. The phenolic compounds and in vitro assays showed antioxidant activity. After gastric ulcer induction by using HCl/ethanol, SpHE reduced the area of ulcerative lesions by 82, 91, and 88%, respectively. In ethanol, SpHE reduced the area of ulcerative lesions by 77, 93, and 92%, respectively. In the NSAID, the percentages of protection were 70, 76, and 78%, respectively. SpHE promoted the minimization of ulcers, increased the levels of reduced glutathione, and decreased tumor necrosis factor. S. purpurea has antioxidant and antiulcer properties.

The role of local disease perception in the selection of medicinal plants: A study of the structure of local medical systems.

ETHNOPHARMACOLOGICAL RELEVANCE: In this study, we investigated the role of local perceptions of diseases in the selection of medicinal plants. For this purpose, we consulted local experts from two communities located in the semiarid region of northeastern Brazil. MATERIALS AND METHODS: After selecting the experts, we used semi-structured interviews to assess the symptoms of each disease studied, the perception of variations in each of these diseases, and the plants used for their treatment. We then conducted a participatory workshop in which the experts engaged in free grouping of the diseases mentioned in the first step. RESULTS: We observed that the therapeutic targets (diseases) showed a lower similarity of medicinal plants than the perceived variants of these targets. We found that plant selection was associated with the perception of symptoms: the greater the similarity between two diseases based on the perceived symptoms, the greater the similarity between these diseases based on the medicinal plants. Furthermore, we observed a greater similarity between plants used to treat diseases perceived as related to each other. CONCLUSION: Local perceptions of the association between diseases and between diseases and symptoms can influence plant selection such that the similarity of the medicinal plants chosen is higher for the diseases perceived as related and as having common symptoms. These results indicate the presence of mechanisms by which local perceptions of diseases affect the structures of local medical systems.

Amebicidal activity of the essential oils of Lippia spp. (Verbenaceae) against Acanthamoeba polyphaga trophozoites.

Amoebic keratitis and granulomatous amoebic encephalitis are caused by some strains of free-living amoebae of the genus Acanthamoeba. In the case of keratitis, one of the greatest problems is the disease recurrence due to the resistance of parasites, especially the cystic forms, to the drugs that are currently used. Some essential oils of plants have been used as potential active agents against this protist. Thus, the aim of this study was to determine the amebicidal activity of essential oils from plants of the genus Lippia against Acanthamoeba polyphaga trophozoites. To that end, 8 × 10(4) trophozoites were exposed for 24 h to increasing concentrations of essential oils from Lippia sidoides, Lippia gracilis, Lippia alba, and Lippia pedunculosa and to their major compounds rotundifolone, carvone, and carvacrol. Nearly all concentrations of oils and compounds showed amebicidal activity. The IC50 values for L. sidoides, L. gracilis L. alba, and L. pedunculosa were found to be 18.19, 10.08, 31.79, and 71.47 µg/mL, respectively. Rotundifolone, carvacrol, and carvone were determined as the major compounds showing IC50 of 18.98, 24.74, and 43.62 µg/mL, respectively. With the exception of oil from L. alba, the other oils evaluated showed low cytotoxicity in the NCI-H292 cell line. Given these results, the oils investigated here are promising sources of compounds for the development of complementary therapy against amoebic keratitis and granulomatous amoebic encephalitis and can also be incorporated into cleaning solutions to increase their amebicidal efficiency.

Expectorant and antioxidant activities of purified fumarprotocetraric acid from Cladonia verticillaris lichen in mice.

UNLABELLED: The lichen Cladonia verticillaris produces bioactive secondary metabolites, such as fumarprotocetraric (FUM) and protocetraric acids. Species of the genus Cladonia demonstrate anti-tumor, anti-inflammatory and antipyretic activities and have been used in folk medicine to treat respiratory diseases (throat irritation, cough, asthma and tuberculosis). The aim of the present study was to evaluate the expectorant and mucolytic activities of fumarprotocetraric acid in albino Swiss mice. FUM was extracted and purified from an acetone extract of C. verticillaris. The phenol red quantification method was used on the bronchoalveolar lavage fluid following the administration of FUM (25, 50 or 100 mg/kg orally or intraduodenally and 12.5, 25 or 50 mg/kg, intraperitoneally) for the evaluation of expectorant activity. Control groups received either saline solution (7.5 mL/kg) or ambroxol (1 mg/kg) through the same administration routes. Antioxidant activity was evaluated using the thiobarbituric acid reactive species assay in mouse lung tissue treated with the FUM at 25, 50 or 100 mg/kg orally, followed by a lipopolysaccharide solution at 1 mg/kg intrapleurally. The same protocol was used for the control groups using either saline solution (7.5 mL/kg, orally) or N-acetylcysteine (20 mg/kg, orally). RESULTS: Orally administered FUM at doses of 25 and 50 mg/kg promoted significantly greater dose-dependent phenol red activity in the bronchoalveolar lavage and expectorant activity in comparison to the controls (p < 0.05). Lipid peroxidation (malondialdehyde equivalent) was reduced by 50% in the lung tissue. CONCLUSION: The results confirm the expectorant and antioxidant properties of fumarprotocetraric acid produced by the lichen C. verticillaris.

Effect of semisolid formulation of persea americana mill (avocado) oil on wound healing in rats.

The aim of this study was to evaluate the wound-healing activity of a semisolid formulation of avocado oil, SSFAO 50%, or avocado oil in natura, on incisional and excisional cutaneous wound models in Wistar rats. An additional objective was to quantify the fatty acids present in avocado oil. On the 14th day, a significant increase was observed in percentage wound contraction and reepithelialization in the groups treated with 50% SSFAO or avocado oil compared to the petroleum jelly control. Anti-inflammatory activity, increase in density of collagen, and tensile strength were observed inSSFAO 50% or avocado oil groups, when compared to control groups. The analysis of the components of avocado oil by gas chromatography detected the majority presence of oleic fatty acid (47.20%), followed by palmitic (23.66%), linoleic (13.46%) docosadienoic (8.88%), palmitoleic (3.58%), linolenic (1.60%), eicosenoic (1.29%), and myristic acids (0.33%). Our results show that avocado oil is a rich source of oleic acid and contains essential fatty acids. When used in natura or in pharmaceutical formulations for topical use, avocado oil can promote increased collagen synthesis and decreased numbers of inflammatory cells during the wound-healing process and may thus be considered a new option for treating skin wounds.

Antimicrobial, antiproliferative and proapoptotic activities of extract, fractions and isolated compounds from the stem of Erythroxylum caatingae plowman.

In the study, we have examined the antitumor and antimicrobial activities of the methanol extract, the fractions, a fraction of total alkaloids and two alkaloids isolated from the stem of Erythroxylum caatingae Plowman. All test fractions, except the hexane fractions, showed antimicrobial activity on gram-positive bacteria and fungi. The acetate: methanol (95:5), acetate, chloroform and hexane fractions show the highest cytotoxicity activity against the NCI-H292, HEp-2 and K562 cell lines using MTT. The absence of hemolysis in the erythrocytes of mice was observed in these fractions and 6ß-Benzoyloxy-3α-(3,4,5- trimethoxybenzoyloxy) tropane (catuabine B). Staining with Annexin V-FITC and JC-1 was used to verify the mechanism of action of the compounds of E. caatingae that showed cytotoxicity less than 30 µg/mL in leukemic cells. After 48 h of incubation, we observed that the acetate: methanol (95:5), acetate, and chloroform fractions, as well as the catuabine B, increased in the number of cells in early apoptosis, from 53.0 to 74.8%. An analysis of the potential of the mitochondrial membrane by incorporation of JC-1 showed that most cells during incubation of the acetate: methanol (95:5) and acetate fractions (63.85 and 59.2%) were stained, suggesting the involvement of an intrinsic pathway of apoptosis.