Nematostatic activity of isoprenylated guanidine alkaloids from Pterogyne nitens and their interaction with acetylcholinesterase.

Although new nematicides have appeared, the demand for new products less toxic and more efficient for the control of plant-parasitic nematodes are still high. Consequently, studies on natural secondary metabolites from plants, to develop new nematicides, have increased. In this work, nineteen extracts from eleven Brazilian plant species were screened for activity against Meloidogyne incognita. Among them, the extracts of Piterogyne nitens showed a potent nematostatic activity. The alkaloid fraction obtained from the ethanol extract of leaves of P. nitens was more active than the coming extract. Due to the promising activity from the alkaloid fraction, three isoprenylated guanidine alkaloids isolated from this fraction, galegine (1), pterogynidine (2), and pterogynine (3) were tested, showing similar activity to the alkaloid fraction, which was comparable to that of the positive control Temik at 250 µg/mL. At lower concentrations (125-50 µg/mL), compound 2 showed to be the most active one. As several nematicides act through inhibition of acetylcholinesterase (AChE), the guanidine alkaloids were also employed in two in vitro AChE assays. In both cases, compound 2 was more active than compounds 1 and 3. Its activity was considered moderated compared to the control (physostigmine). Compound 2 was selected for an in silico study with the electric eel (Electrophorus electricus) AChE, showing to bind mostly to the same site of physostigmine in the AChEs, pointing out that this could be the mechanism of action for this compound. These results suggested that the guanidine alkaloids 1,2 and 3 from P. nitens are promising for the development of new products to control M. incognita, especially guanidine 2, and encourage new investigations to confirm the mechanism of action, as well as to determine the structure-activity relationship of the guanidine alkaloids.

Targeted Isolation of Indolopyridoquinazoline Alkaloids from Conchocarpus fontanesianus Based on Molecular Networks.

A dichloromethane-soluble fraction of the stem bark of Conchocarpus fontanesianus showed antifungal activity against Candida albicans in a bioautography assay. Off-line high-pressure liquid chromatography activity-based profiling of this extract enabled a precise localization of the compounds responsible for the antifungal activity that were isolated and identified as the known compounds flindersine (17) and 8-methoxyflindersine (18). As well as the identification of the bioactive principles, the ultra-high-pressure liquid chromatography-high-resolution mass spectrometry metabolite profiling of the dichloromethane stem bark fraction allowed the detection of more than 1000 components. Some of these could be assigned putatively to secondary metabolites previously isolated from the family Rutaceae. Generation of a molecular network based on MS(2) spectra indicated the presence of indolopyridoquinazoline alkaloids and related scaffolds. Efficient targeted isolation of these compounds was performed by geometric transfer of the analytical high-pressure liquid chromatography profiling conditions to preparative medium-pressure liquid chromatography. This yielded six new indolopyridoquinazoline alkaloids (5, 16, 19-22) that were assigned structurally. The medium-pressure liquid chromatography separations afforded additionally 16 other compounds. This work has demonstrated the usefulness of molecular networks to target the isolation of new natural products and the value of this approach for dereplication. A detailed analysis of the constituents of the stem bark of C. fontanesianus was conducted.

Trypanocidal activity of Brazilian plants against epimastigote forms from Y and Bolivia strains of Trypanosoma cruzi

Chagas disease is one of the main public health problems in Latin America. Since the available treatments for this disease are not effective in providing cure, the screening of potential antiprotozoal agents is essential, mainly of those obtained from natural sources. This study aimed to provide an evaluation of the trypanocidal activity of 92 ethanol extracts from species belonging to the families Annonaceae, Apiaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Moraceae, Nyctaginaceae, and Verbenaceae against the Y and Bolivia strains of Trypanosoma cruzi. Additionally, cytotoxic activity on LLCMK2 fibroblasts was evaluated. Both the trypanocidal activity and cytotoxicity were evaluated using the MTT method, in the following concentrations: 500, 350, 250, and 100 µg/mL. Benznidazole was used for positive control. The best results among the 92 samples evaluated were obtained with ethanol extracts of Ocotea paranapiacabensis (Am93) and Aegiphila lhotzkiana (Am160). Am93 showed trypanocidal activity against epimastigote forms of the Bolivia strain and was moderately toxic to LLCMK2 cells, its Selectivity Index (SI) being 14.56, while Am160 showed moderate trypanocidal activity against the Bolivia strain and moderate toxicicity, its SI being equal to 1.15. The screening of Brazilian plants has indicated the potential effect of ethanol extracts obtained from Ocotea paranapiacabensis and Aegiphila lhotzkiana against Chagas disease.

Antifungal activity of Piper diospyrifolium Kunth (Piperaceae) essential oil

In vitro activity of the essential oil from Piper diospyrifolium leaves was tested using disk diffusion techniques. The antifungal assay showed significant potencial antifungal activity: the oil was effective against several clinical fungal strains. The majority compounds in the essential oil were identified as sesquiterpenoids by GC-MS and GC-FID techniques.

Interconverting flavanone glucosides and other phenolic compounds in Lippia salviaefolia Cham. ethanol extracts.

Four interconverting flavanone glycosides [(2R)- and (2S)-3',4',5,6-tetrahydroxyflavanone 7-O-ß-D-glucopyranoside, and (2R)- and (2S)-3',4',5,8-tetrahydroxyflavanone 7-O-ß-D-glucopyranoside], in addition to eight known flavonoids [naringenin, asebogenin, sakuranetin, 6-hydroxyluteolin 7-O-ß-D-glucoside, (2R)- and (2S)-eriodictyol 7-O-ß-D-glucopyranoside, aromadendrin and phloretin], three phenylpropanoid glycosides [forsythoside B, alyssonoside and verbascoside] and the epoxylignan lariciresinol 4'-O-ß-D-glucopyranoside were isolated and identified in the EtOH extract of the aerial parts of Lippia salviaefolia Cham. The phytochemical study herein was guided by preliminary antioxidant tests, namely, ß-carotene protection and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity. The crude extracts, their active fractions and the isolated compounds were assayed against intracellular reactive oxygen species (ROS) and human embryonic kidney HEK-293 and human melanoma M14 cancer cell growth. Aromadendrin and phloretin were able to counteract elevation of ROS induced by the oxidant t-butylhydroperoxide (t-BOOH) in HEK-293 cells, whereas phloretin strongly protected HEK-293 cells from ROS damage at 1 µM. Additionally, phloretin exhibited a significant growth inhibitory effect at 20-40 µM in both HEK-293 and M14 cells and induced a concentration dependent apoptosis at 20 µM in M14 cells, suggesting a selective action towards malignant cells. Due to their equilibria, the four interconverting flavanone glycosides were studied using 1D and 2D NMR, HPLC-CD-PDA and HRMS analyses.

Antifungal activity of Piper diospyrifolium Kunth (Piperaceae) essential oil.

In vitro activity of the essential oil from Piper diospyrifolium leaves was tested using disk diffusion techniques. The antifungal assay showed significant potencial antifungal activity: the oil was effective against several clinical fungal strains. The majority compounds in the essential oil were identified as sesquiterpenoids by GC-MS and GC-FID techniques.

Alkaloids from stems of Esenbeckia leiocarpa Engl. (Rutaceae) as potential treatment for Alzheimer disease.

Esenbeckia leiocarpa Engl. (Rutaceae), popularly known as guarantã, goiabeira, is a native tree from Brazil. Bioactivity-guided fractionation of the ethanol stems extract afforded the isolation of six alkaloids: leiokinine A, leptomerine, kokusaginine, skimmianine, maculine and flindersiamine. All isolated compounds were tested for acetyl cholinesterase inhibition, in vitro and displayed anticholinesterasic activity. The alkaloid leptomerine showed the highest activity (IC50 = 2.5 mM), similar to that of the reference compound galanthamine (IC50 = 1.7 mM). The results showed for the first time the presence of alkaloids leptomerine and skimmianine in E. leiocarpa (Engl.) with potent anticholinesterasic activity.

Trypanocidal, leishmanicidal and antifungal potential from marine red alga Bostrychia tenella J. Agardh (Rhodomelaceae, Ceramiales).

Specimens of the red alga Bostrychia tenella J. Agardh (Rhodomelaceae, Ceramiales) were collected from the São Paulo coast and submitted to room temperature solvent extraction. The resulting extract was fractionated by partitioning with organic solvent. The n-hexane (BT-H) and dichloromethane (BT-D) fractions showed antiprotozoal potential in biological tests with Trypanosoma cruzi and Leishmania amazonensis and presented high activity in an antifungal assay with the phytopathogenic fungi Cladosporium cladosporioides and Cladosporium sphaerospermum. Chromatography methods were used to generate subfractions from BT-H (H01 to H11) and from BT-D (D01 to D19). The subfractions were analyzed by gas chromatography-mass spectrometry (GC/MS), and the substances were identified by retention index (Kovats) and by comparison to databases of commercial mass spectra. The volatile compounds found in marine algae were identified as fatty acids, low molecular mass hydrocarbons, esters and steroids; some of these have been previously described in the literature based on other biological activities. Moreover, uncommon substances, such as neophytadiene were also identified. In a trypanocidal assay, fractions BT-H and BT-D showed IC(50) values of 16.8 and 19.1 microg/mL, respectively, and were more active than the gentian violet standard (31 microg/mL); subfractions H02, H03, D01 and D02 were active against L. amasonensis, exhibiting IC(50) values of 1.5, 2.7, 4.4, and 4.3 microg/mL, respectively (standard amphotericin B: IC(50)=13 microg/mL). All fractions showed antifungal potential. This work reports the biological activity and identification of compounds by GC/MS for the marine red alga B. tenella for the first time.

Isolamento e avaliação da atividade biológica dos alcalóides majoritários de Tabernaemontana angulata Mart. ex Müll. Arg., Apocynaceae / Isolation and evaluation of the biological activity related to the major alkaloids in Tabernaemontana angulata Mart. ex Müll. Arg., Apocynaceae

A necessidade da introdução de novos agentes quimioterápicos é uma realidade no controle de doenças infecciosas. O Brasil é o país mais rico em biodiversidade, e o Laboratório de Extração da Universidade Paulista tem coletado plantas na Amazônia e Mata Atlântica com a finalidade de identificar extratos vegetais antibacterianos e antitumorais. Estudos prévios demonstraram que a fração etanólica obtida do extrato bruto orgânico do caule de Tabernaemontana angulata Mart. ex Müll. Arg. apresentou atividade antimicrobiana significante contra Staphylococcus aureus ATCC6538, no ensaio de diluição em caldo. Verificou-se por cromatografia em camada delgada que os compostos majoritários presentes na fração ativa eram alcalóides. No presente trabalho, foi obtida a fração de alcalóides totais a partir do extrato bruto, da qual os compostos majoritários foram isolados por sucessivas cromatografias e, posteriormente, identificados por CG-EM e ¹H-RMN como os alcalóides indólicos coronaridina e voacangina. As frações obtidas do isolamento foram testadas no ensaio biológico, porém não demonstraram atividade antimicrobiana.

Introducing new chemotherapeutic agents is a great demand in the control of infectious diseases. Brazil is one of the richest countries in biodiversity and the Laboratório de Extração at UNIP has been collecting plants from the Amazon and Atlantic Rain Forests with the aim of screening for new antibacterial and antitumor plant extracts. Previous studies demonstrated that the ethanol fraction obtained from the crude extract of Tabernaemontana angulata stems showed an antibacterial activity against Staphylococcus aureus (ATCC 6538) in the microdilution broth assay. Two alkaloids were the major compounds in the active fraction, verified by thin layer chromatography analysis. In the present study, the total alkaloids were obtained from the crude extract and were fractionated by preparative thin layer chromatography for the isolation of the main components. The isolated compounds were identified by GC/MS and ¹H-NMR as coronaridine and voacangine. The alkaloid fractions obtained from the isolation procedure were tested for antibacterial activity, but no activity was detected.

Cytotoxic guanidine alkaloids from Pterogyne nitens.

As part of a bioprospecting program aimed at the discovery of potential anticancer drugs, two new guanidine-type alkaloids, nitensidines D and E (1, 2), and the known pterogynine (3), pterogynidine (4), and galegine (5), were isolated from the leaves of Pterogyne nitens. The structures of 1 and 2 were established on the basis of spectroscopic data interpretation. These compounds were tested against a small panel of human cancer cell lines. Compound 2 exhibited cytotoxicity for HL-60 (human myeloblastic leukemia) and SF-245 (human glioblastoma) cells.

Screening for antifungal, DNA-damaging and anticholinesterasic activities of Brazilian plants from the Atlantic Rainforest: Ilha do Cardoso State Park / Avaliação das atividades antifúngica, no reparo do DNA e anticolinesterásica de plantas brasileiras da Mata Atlântica: Parque Estadual da Ilha do Cardoso

Crude extracts from 17 plant species collected from an Atlantic Forest regionin the State of São Paulo (Brazil) have been screened for antifungal, DNA-damaging and acetylcholinesterase inhibiting activities. Of the 34 extracts obtained from leaves and stems ofplants assayed for antifungal activity with Cladosporium sphaerospermum and C. cladosporioides 26.5% were active. However, only the extract of leaves of Cabralea canjerana showed a stronginhibition of both fungi. The DNA-damaging assay with mutant strains of Saccharomyces cerevisiae resulted in 11.7 % of the extracts being active whereas 100% of them showedselectivity for the DNA-repair mechanism of topoisomerase II. Of the 17 species analysed, 12 showed anticholinesterasic activity in TLC assay. However, only extracts from Tetrastylidium grandifolium (stems) and Sloanea guianensis (leaves and stems) inhibited acetylcholinesterase activity more than 50% in quantitative assay.

Extratos brutos de 17 espécies de plantas coletadas em região de Mata Atlântica no Estado de São Paulo (Brasil)foram avaliadas para as atividades: antifúngica, no reparo do DNA e inibição da acetilcolinesterase. Dos 34 extratos obtidos de folhas e galhos das plantas analisadas para a atividade antifúngicacom Cladosporium sphaerospermum e C. cladosporioides, 26,5% foram ativos. Todavia, apenas o extrato das folhas de Cabralea canjerana inibiu fortemente o crescimento dos dois fungos. No ensaio de reparo do DNA com linhagens mutantes de Saccharomyces cerevisiae, 11,7% dos extratos foram ativos, sendo que, 100% destes foram seletivos para o mecanismo de reparo do DNA envolvendo topoisomerase II. Das 17 espécies analisadas, 12 demonstraram atividade anticolinesterásica no ensaio qualitativo sobre cromatografi a de camada delgada (CCD). No entanto, apenas os extratos de Tetrastylidium grandifolium (galhos) e Sloanea guianensis (folhas e galhos) apresentaram atividade anticolinesterásica maior que 50% no ensaio quantitativo.

Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors.

A myeloperoxidase inhibitory kaempferol derivative, namely pterogynoside (1), was isolated from fruits of Pterogyne nitens, along with six known flavonols, kaempferol, afzelin, kaempferitrin, quercetin, isoquercetrin and rutin. The structures of all compounds were elucidated primarily from 1D and 2D NMR spectroscopic analyses, as well as by high resolution mass spectrometry. All flavonols were screened to identify secondary metabolites as potential myeloperoxidase (MPO) inhibitors, and at concentrations of 0.50-50nM, quercetin (5), isoquercitrin (6) and rutin (7) exhibited strong inhibitory effects with IC50 values of 1.22+/-0.01, 3.75+/-0.02 and 3.60+/-0.02, respectively. The MPO activity detected for the new derivative 1 was markedly decreased (IC(50) 10.3+/-0.03) when compared with known flavonols 5-7, and interestingly increased when tested against ABTS scavenging activity.

Bioactive flavone dimers from Ouratea multiflora (Ochnaceae)

Chromatographic fractionation of the organic extract from leaves of Ouratea multiflora afforded the flavone dimers heveaflavone, amentoflavone-7'',4''''-dimethyl eter, podocarpusflavone-A and amentoflavone. Their structures were elucidated from spectral data, including 2D-NMR experiments of the natural substances. Biological activities of all isolates were evaluated, using antimicrobial assay against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, cytotoxicity assay against mouse lymphoma (L5178) and KB cell lines, TLC screening for acetylcholinesterase inhibitors and antioxidant activity measured by DPPH test.

O fracionamento cromatográfico do extrato orgânico das folhas de Ouratea multiflora forneceu os flavonóides diméricos, heveaflavona, 7'',4''''-dimetilamentoflavona, podocarpusflavona-A e amentoflavona. Suas estruturas foram elucidadas com base nos dados espectrais, incluindo experimentos bidimensionais de RMN, das substâncias naturais. A atividade antibiótica de todos os isolados foi avaliada, usando-se as bacterias Gram-positivas Staphylococcus aureus and Bacillus subtilis. Teste de citotoxicidade nas linhagens de linfoma de ratos (L5178) e KB também foram conduzidos para avaliar os extratos e os flavonóides isolados. a triagem biológica para a avaliação de atividade antioxidante e inibidora de acetil colinesterase foram conduzidas pela técnica da bioautografia com DPPH e teste pelo teste de Ellman respectivamente.

Oleanane and taraxerane glycosides from the roots of Gomphrena macrocephala.

Phytochemical screening of the roots of Gomphrena macrocephala, with particular attention to its triterpene glycoside constituents, has resulted in the isolation of two new oleanane glycosides (1 and 2) and a new taraxerane glycoside (3). The structures of 1-3 were determined as 11alpha,12alpha-epoxy-3beta-[(O-beta-D-glucuronopyranosyl)oxy]olean-28,13-olide (1), 11alpha,12alpha-epoxy-3beta-[(O-beta-D-galactopyranosyl-(1-->3)-O-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl)-oxy]olean-28,13-olide (2), and 11alpha,12alpha-epoxy-3beta-[(O-beta-D-glucuronopyranosyl)oxy]taraxer-14-en-28-oic acid beta-D-glucopyranosyl ester (3), respectively, on the basis of their spectroscopic data and the results of hydrolysis. The aglycones (1a and 3a) of 1-3 with an epoxy group showed cytotoxic activity against HSC-2 human oral squamous carcinoma cells.

Aromatic compounds produced by Periconia atropurpurea, an endophytic fungus associated with Xylopia aromatica.

6,8-Dimethoxy-3-(2'-oxo-propyl)-coumarin (1) and 2,4-dihydroxy-6-[(1'E,3'E)-penta-1',3'-dienyl]-benzaldehyde (2), in addition to the known compound periconicin B (3), were isolated from the ethyl acetate extract of Periconia atropurpurea, an endophytic fungus obtained from the leaves of Xylopia aromatica, a native plant of the Brazilian Cerrado. Their chemical structures were assigned based on analyses of MS, 1D and 2D-NMR spectroscopic experiments. Biological analyses were performed using two mammalian cell lines, human cervix carcinoma (HeLa) and Chinese hamster ovary (CHO). The results showed that compound 1 had no effect when compared to the control group, which was treated with the vehicle (DMSO). Compound 2 was able to induce a slight increase in cell proliferation of HeLa (37% of increase) and CHO (38% of increase) cell lines. Analysis of compound 3 showed that it has potent cytotoxic activity against both cell lines, with an IC50 of 8.0 microM. Biological analyses using the phytopathogenic fungi Cladosporium sphaerospermum and C. cladosporioides revealed that also 2 showed potent antifungal activity compared to nystatin.

Phenylpropanoid glucosides from leaves of Coussarea hydrangeifolia (Rubiaceae).

Phenylpropanoid glycosides, 1'-O-benzyl-alpha-L-rhamnopyranosyl-(1''-->6')-beta-D-glucopyranoside (1) and alpha-L-xylopyranosyl-(4''-->2')-(3-O-beta-D-glucopyranosyl)-1'-O-E-caffeoyl-beta-D-glucopyranoside (2), together with the known derivatives, 1,6-di-O-caffeoyl-beta-D-glucopyranoside (3), 1-O-(E)-caffeoyl-beta-D-glucopyranoside (4) and 1-O-(E)-feruloyl-beta-D-glucopyranoside (5), were isolated from leaves of Coussarea hydrangeifolia. Their structures were determined by IR, HRESIMS, and 1D and 2D NMR experiments, and their antioxidant activities, evaluated by assaying the free radical scavenging capacity using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical as substrate. The antioxidant activities of 3 and 4 (IC50 values of 15.0 and 19.2 microM, respectively) were comparable to that of the standard positive control caffeic acid, whilst 2 and 5 were only weakly active and 1 was inactive.

Benzopyrans from Curvularia sp., an endophytic fungus associated with Ocotea corymbosa (Lauraceae).

An isolate of Curvularia sp. was obtained from the leaves of Ocotea corymbosa, a native plant of the Brazilian Cerrado. The ethyl acetate extract from culture of this fungus afforded two benzopyran derivatives: (2'S)-2-(propan-2'-ol)-5-hydroxy-benzopyran-4-one (2) and 2,3-dihydro-2-methyl-benzopyran-4,5-diol (4); and two known benzopyrans: 2-methyl-5-methoxy-benzopyran-4-one (1) and (2R)-2,3-dihydro-2-methyl-5-methoxy-benzopyran-4-one (3). The structures of 2 and 4 were established on the basis of comprehensive spectroscopic analysis, mainly using 1D and 2D NMR experiments. The benzopyrans 1 and 2 showed weak in vitro antifungal activity against Cladosporium sphaerospermum and C. cladosporioides. Analyses of the biological activities were also carried out on HeLa (human cervix tumor) and CHO (Chinese hamster ovary) cells, aiming to evaluate their potential effects on mammalian cell line proliferation. Results from both cell lines indicated that compound 2 was able to induce cell proliferation: 70% on HeLa cells and 25% on CHO cells.

New antioxidant C-glucosylxanthones from the stems of Arrabidaea samydoides.

Three new C-glucosylxanthones, 2-(2'-O-trans-caffeoyl)-C-beta-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (1), 2-(2'-O-trans-cinnamoyl)-C-beta-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (2), and 2-(2'-O-trans-coumaroyl)-C-beta-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (3), were isolated from the stems of Arrabidaea samydoides, in addition to three known C-glucosylxanthones, mangiferin (4), 2-(2'-O-benzoyl)-C-beta-d-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (5), and muraxanthone (6). Their chemical structures were assigned on the basis of MS and 1D and 2D NMR experiments. Xanthones 1-6 showed moderate free radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) as well as antioxidant activity evidenced by redox properties measured on ElCD-HPLC.

Antibacterial xanthones from Kielmeyera variabilis mart. (Clusiaceae).

The bioassay-guided fractionation of stems from Kielmeyera variabilis, traditionally used in Brazilian folk medicine, yielded assiguxanthone-B (1), kielcorin (4), 2,5-dihydroxybenzoic acid (3), and a mixture of xanthones containing assiguxanthone-B (1) and 1,3,5,6-tetrahydroxy-2-prenylxanthone (2) (1:1 w/w). The xanthone mixture inhibited Staphylococcus aureus and Bacillus subtilis at a concentration of 6.25 g/ml. When tested alone, the minimal inhibitory concentration of assiguxanthone-B was 25 g/ml against B. subtilis. Kielcorin and 2,5-dihydroxybenzoic acid were inactive against both strains. None of the fractions was active against Escherichia coli or Pseudomonas aeruginosa. Viable cells of S. aureus were reduced by a 1-3 log CFU/ml within 12 h after exposure of one to eight times the MIC of the xanthone mixture. It is not known whether the tetrahydroxy-2-prenylxanthone or other components of the xanthone mixture are responsible for the main antibacterial activity or whether additive or synergistic action is involved

Turbinatine, a potential key intermediate in the biosynthesis of corynanthean-type indole alkaloids.

Extraction of the leaves of Chimarrhis turbinata has led to the isolation of turbinatine (1), a new corynanthean-type indole alkaloid, besides four known indole alkaloids, strictosidine, 5alpha-carboxystrictosidine, vallesiachotamine, and isovallesiachotamine. The structural determination of 1 was based on 1D and 2D spectroscopic data. An evaluation of the DNA-damaging activities of the isolates was performed by means of a bioassay using mutant strains of Saccharomyces cerevisiae, which indicated these compounds were weakly active.