AntiHepatitis C Virus Activity of Alectryon serratus Leaves Extract

Hepatitis C Virus (HCV) has infected approximately 2-3% (130-170 million) of the world's population. No vaccine is available to prevent HCV infection. Investigation of anti-HCV agent is thus deemed necessary. Various plants have been explored for their anti-HCV activity. A. serratus is a member of Sapindaceae family, which fruit and seed were traditionally used as insecticide. Anti-HCV activity tested on A.serratus leaves extract has been done. The result showed that leaves extract exhibited anti-HCV with IC₅₀ value of 14.9 μg/ml and 9.8 μg/ml against HCV J6/JFH1 and JFH1a, respectively. The cytotoxicity assay results showed that A.serratus leaves extract was not toxic and has CC₅₀ >100 μg/ml. Mode of action experiment results suggested that A.serratus extract inhibited HCV at the post-entry step. Further fractionation of leaves extract by open column chromatography resulted in 4 fractions. Only Fraction 1 (AP-5F.1) exhibited anti-HCV with IC₅₀ value of 1.2 μg/ml against HCV JFH1a. Separation of AP-5F.1 by open column chromatography resulted in 15 fractions. Fraction number 13 (AP-5F.1.13) exhibited anti-HCV with IC₅₀ value of 0.43 μg/ml against HCV JFH1a. Separation of AP-5F.1.13 by semi preparative-HPLC resulted in isolate identified by TLC and LC-MS method as chlorophyll derivate. There was a possibility that chlorophyll derivate has participated in performing the anti-HCV activity of fractions and extract besides the other compounds contained. In this study, we concluded that A. serratus leaves extract, AP-5F.1, and AP-5F.1.13 exhibited anti-HCV activity against JFH1a virus.     

Antioxidant and α-Glucosidase Inhibitory Compounds of Centella Asiatica

Centella asiatica, as known as Pegagan was previously reported to have anti-hyperglycemic effects in animal diabetic model rats. However, its α-glucosidase activity in vitro assay not yet reported. Our goal in this study is to isolate and identify active compounds as α-glucosidase inhibitor and antioxidant from aqueous ethanol 70% (v/v) extract of C. asiatica. The extract was partitioned by n-hexane, EtOAc, and n-butanol sequentially. Among the fractions tested, EtOAc fraction was showed the highest antioxidant and α-glucosidase inhibitory activities with an IC₅₀ values of 45.42 and 73.17 μg/mL, respectively. The antioxidant activity was conducted by determination of DPPH radical scavenging activity, whereas α-glucosidase inhibitory activity was determined against yeast α-glucosidase. Furthermore, isolation of the ethyl acetate extract yielded two active compounds, which were identified as kaempferol (1) and quercetin (2). Both of the compounds showed good yeast α-glucosidase inhibitory activity with IC₅₀ values of 16.50 and 21.61 μg/mL, respectively. In addition those compounds also could scavenge DPPH radical activity with IC₅₀ values of 9.64 and 11.97 μg/mL, respectively. Due to its ability in reducing α-glucosidase activity and scavenging free radical activity, the C. asiatica appears to be a potential as a good resource for future development of antioxidant and antidiabetic drug.     

Ruellia prostrata Poir. activity evaluated by phytoconstituents,antioxidant, anti-inflammatory,antibacterial activity,and in silico molecular functions

Ruellia prostrata Poir. has been used historically as an anti-cancer, wound healing agent and to treat gonorrhea. We aimed to determine the phytochemicals present in ethyl acetate extract of R. prostrata Poir. (EAERP). We sought to determine the antioxidant, anti-inflammatory, and antibacterial activities in vitro, and toxicity properties in vivo. We also analyzed the Prediction of Activity Spectra for Substances (PASS), physicochemical, ADMET, and drug-likeness properties of phytochemicals in EAERP. To determine phytoconstituents, preliminary phytochemical screening and GC–MS were performed, while FT-IR was used to identify functional groups. The antioxidant activity was evaluated using a DPPH scavenging assay, whereas BSA denaturation and RBC hemolysis inhibition were used to assess anti-inflammatory activity. An agar-well-diffusion assay was performed to estimate the antibacterial activity. Brine shrimp lethality bioassay and oral delivery of EAERP of single-dose were performed to determine cytotoxicity and acute toxicity, respectively. The phytochemical screening revealed the presence of phenols, triterpenoids, saponins, steroids, amino acids, and fat and fixed oils. FT-IR analysis of EAERP showed the presence of many functional groups: alcohols/phenols, carboxylic acids, aldehydes, alkanes, esters, amines, amides, aromatic hydrocarbons, sulfoxides, and alkyl halides. GC–MS revealed the presence of 39 phytoconstituents including steroids, consistent with compounds and functional groups found in preliminary screening and FT-IR. EAERP showed dose-dependent antioxidant activity with an IC₅₀ value of 21.402 µg/mL and anti-inflammatory activity with an IC₅₀ value of 20.564 µg/mL in RBC hemolysis inhibition and 21.115 µg/mL in BSA denaturation assays. EAERP also exhibited dose-related antibacterial activity. EAERP exerted cytotoxicity with an LC₅₀ value of 17.619 μg/mL and acute toxicity with an LD₅₀ value of 4095.328 mg/kg without any adverse effects. The PASS server also predicted that the phytoconstituents of EAERP have antioxidant, anti-inflammatory, and antibacterial activities with probable activity (Pa) ranging from 0.310 to 0.717. Analysis of physicochemical, ADMET, and drug-likeness properties revealed the drug-able efficacy and safety of most compounds. The findings of this study indicated that R. prostrata Poir. contains phytoconstituents with potent antioxidant, anti-inflammatory, and antibacterial activities. Taken together, our measurements suggest that R. prostrata Poir. is a prime candidate for further exploration as a potential therapeutic agent.     

Synthesis,antiviral activity,and 3D-QSAR study of novel chalcone derivatives containing malonate and pyridine moieties

Several novel chalcone derivatives containing malonate and pyridine moieties were synthesized, and their structures were confirmed by ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance, ¹⁹F nuclear magnetic resonance, infrared, and elemental analyses. Antiviral bioassays revealed that most of the compounds exhibited good antiviral activity against cucumber mosaic virus (CMV) at 500 μg/mL. In particular, compounds 5l and 5n showed significant curative activities against CMV in vivo with 50% effective concentration (EC₅₀) values of 186.2 and 211.5 μg/mL, respectively; these values are even better than that of ningnanmycin (330.5 μg/mL). A 3D quantitative structure–activity relationship study was carried out using the comparative molecular field analysis technique based on curative activities against CMV. Results revealed good predictive ability with cross-validated q² and non-cross-validated r² values of 0.517 and 0.990, respectively.     

Synthesis and anti-inflammatory and antimicrobial activities of some novel 2-methylquinazolin-4(3H)-one derivatives bearing urea,thiourea and sulphonamide functionalities

A variety of novel 2-methylquinazolin-4(3H)-one derivatives (1–29) bearing urea, thiourea and sulphonamide functionalities at position 3 of biological interest have been synthesized and screened for their anti-inflammatory (TNF-α and IL-6) and antimicrobial activities (antibacterial and antifungal). “Biological evaluation study revealed that the compounds 3, 4, 6, 9, 16 and 18 were found to have promising anti-inflammatory activity (up to 62–84% TNF-α and 73–92% IL-6 inhibitory activity) at concentration of 10 μM with reference to standard dexamethasone (75% TNF-α and 84% IL-6 inhibitory activities at 1 μM) and 7, 10, 12, 23, 25 and 28 have antimicrobial activity at MIC of 10–30 μg/mL against selected pathogenic bacteria and fungi.”     

Synthesis of some Mannich base derivatives and their antimicrobial activity study

A series of 2-(phenyl)-2-(morpholin-4-yl)-N-phenylacetamide I–VII were synthesized by Mannich base method. Synthesized compounds I–VII were confirmed by IR, ¹H NMR, ¹³C NMR, mass and elemental analyses. Synthesized compounds I–VII were screened for antibacterial activity against various bacterial strains and compared with standard Ciprofloxacin at concentration 100 μg/mL and for antifungal activity against various fungal strains and compared with Clotrimazole at concentration 100 μg/mL; particularly 3-(4-chlorophenyl)-3-(morp holin-4-yl)-N-phenylpropanamide lll that has high antibacterial activity against Streptococcus epidermidis was compared with standard Ciprofloxacin.     

Anti-inflammatory and antibacterial activity study of some novel quinazolinones

Anti-inflammatory and antibacterial activities of some novel quinazolinones were determined. Evaluation of anti-inflammatory activity of test compounds was performed using carrageenan induced paw edema in rats. Oral administration of test compounds 25 mg/kg and 50 mg/kg reduced the paw edema significantly (P < 0.05) in a dose dependent manner compared to carrageenan induced rats. The test compounds were also screened for their antibacterial activity against the strains of Staphylococcus aureus and Escherichia coli at the concentrations of 200 μg/ml and 1 mg/ml. The test compounds showed better activity as that of the standard lincomycin at the tested higher concentration against S. aureus. None of the compounds exhibit comparable activity to that of the standard ceftazidime against E. coli.     

Synthesis,biological evaluation and Structure Activity Relationships (SARs) study of 8-(substituted)aryloxycaffeine

A series of 8-(substituted)aryloxycaffeine were prepared from 8-bromocaffeine and (substituted)phenols by modified Ullmann reaction. In vitro antibacterial activity, inhibitory activity on topoisomerase II and pharmacological activities were evaluated for the synthesized 8-(substituted)aryloxycaffeine. Among the synthesized compounds, 8-(5-chloropyridin-3-yloxy)caffeine (3k) showed strong inhibitory activity (MIC = 15.6 μg/mL) against the tested gram negative (−) bacteria Salmonella enteritidis. 8-(quinolin-8-yloxy)caffeine (3g) showed the strongest inhibitory activity against topoisomerase II. And the compounds 8-(6-methylpyridin-2-yloxy)caffeine (3j) and 8-(3-chloro-6-(trifluoromethyl)pyridin-2-yloxy)caffeine (3m) showed analgesic effect without the central nervous system stimulation.     

Synthesis and antiviral bioactivity of novel chalcone derivatives containing purine moiety

A series of novel chalcone derivatives containing purine group was synthesized and evaluated for their antiviral activities against cucumber mosaic virus and tobacco mosaic virus. Compound 3o exhibited remarkable antiviral activities and strong combining capacity to tobacco mosaic virus coat protein.     

The role of gamma irradiation on the extraction of phenolic compounds in onion (Allium cepa L.)

The effect of gamma irradiation on the content of total phenolic compounds, especially quercetin (Q), in onion (Allium cepa L.) skin was investigated. Onion skin extracts contained two predominant flavonoid compounds, Q and quercetin-4′-glucoside (Q4′G). After 10 kGy gamma irradiation, the yield of Q in the extracts increased significantly from 36.8 to 153.9 μg/ml of the extract, and the Q4′G content decreased slightly from 165.0 to 134.1 μg/ml. In addition, the total phenolic compound content also increased after irradiation at 10 kGy, from 228.0 μg/g of fresh weight to 346.6 μg/g; negligible changes (237.1–256.7 μg/g) occurred at doses of up to 5 kGy. As we expected, radical-scavenging activity was enhanced remarkably (by 88.8%) in the 10 kGy irradiated sample. A dose-dependent increase in the peak intensity of the electron paramagnetic resonance (EPR) spectra was observed in all irradiated samples, with a maximum increase at 10 kGy. The intensity relative to that of the control was 0.15, and it increased to 1.10 in 10 kGy irradiated samples. The optimum gamma irradiation dose, which is sufficient to break the chemical or physical bonds and release soluble phenols of low molecular weight in onion skin, is about 10 kGy.     

Synthesis,characterization, X-ray diffraction,antimicrobial and in vitro cytotoxicity studies of 7a-Aza-B-homostigmast-5-eno [7a,7-d] tetrazole

The synthesis, spectral characterization, crystal structure and antimicrobial activity of the novel synthetic molecule 7a-Aza-B-homostigmast-5-eno [7a,7-d] tetrazole, C₂₉H₄₈N₄ has been reported. The structure has also been determined by X-ray diffraction technique using direct method and was refined on F² by the full-matrix least-squares. Crystals are orthorhombic and their space group is P212121, with a = 7.230(3), b = 31.451(13), c = 11.974(5) (Å), α = β = γ = 90°. It can be conveniently obtained by the reaction of 7-Oxostigmast-5-ene with hydrazoic acid. The molecule has also been screened for its possible in vitro antimicrobial activity against Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes, Staphylococcus epidermidis, Bacillus cereus, Corynebacterium xerosis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa (MTCC 424). Minimum inhibitory concentration (MIC) of the synthesized compound has also been evaluated. The highest activity is observed against C. xerosi and P. vulgaris. Moreover, the compound has also been screened for its in vitro cytotoxicity against human colon carcinoma cell line, HCT116 and human liver hepatocellular carcinoma cell line, HepG2, using doxorubicin as standard. On the basis of its IC₅₀ values, 7a-Aza-B-homostigmast-5-eno [7a,7-d] tetrazole was found to inhibit the cancer cells effectively.     

Anti-Cancer Effect of Kaffir Lime (Citrus Hystrix DC) Leaf Extract in Cervical Cancer and Neuroblastoma Cell Lines

Previous study showed that kaffir lime leaf contains alkaloid, flavonoid, terpenoid, tannin and saponin. The objective of this study was to examine the cytotoxic effect of kaffir lime leaf extract on cervical cancer and neuroblastoma cell lines. The method used for this research to determine cell viability was an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results showed that an ethyl acetate extract had an IC50 for HeLa cells, UKF-NB3, IMR-5 and SK-N-AS parental cells of 40.7 μg · mL^–1, 28.4 μg · mL^–1, 14.1 μg · mL^–1, and 25.2 μg · mL^–1 respectively. Furthermore, the IC50 of chloroform extracts for HeLa cells, UKF-NB3, IMR-5 and SK-N-AS parental were 17.6 μg · mL^–1, 18.9 μg · mL^–1, 6.4 μg · mL^–1, and 9.4 μg · mL^–1 respectively. These data showed that kaffir lime extract reduces the viability of cervical and neuroblastoma cell lines and may have potential as anti-cancer compounds.     

HPLC–DAD–MS identification of polyphenols from Passiflora leschenaultii and determination of their antioxidant,analgesic, anti-inflammatory and antipyretic properties

Passion fruit (Passiflora leschenaultii DC), an endemic species to peninsular India, is traditionally used to treat various ailments such as dysentery, urinary stone disease and wounds. The present study aimed to investigate the antioxidant, analgesic, anti-inflammatory, antipyretic activities and chemical composition of leaf extracts of P. leschenaultii. Bioactive secondary metabolites such as total phenolics, tannins and flavonoids were quantified. Antioxidant activities were determined by DPPH, ABTS⁺, FRAP, metal chelating and phosphomolybdenum assays. Hot plate, acetic acid and formalin induced pain models were used to evaluate the analgesic activity. In order to study the acute and chronic anti-inflammatory activities, carrageenan and cotton pellet induced models were used in rats. Brewer’s yeast induced pyrexia method was applied for the antipyretic test. Functional compounds from the plant were identified and quantified through HPLC–DAD–MS analysis. The obtained results revealed that the acetone extract of leaves exhibited higher phenolic (440.24 mg GAE/g extract) and flavonoid (253.33 mg RE/g extract) contents and scavenged the DPPH (IC₅₀ 29.14 μg/mL), ABTS⁺ (10509.69 μM TEAC/g extract) effectively. On investigating the analgesic, anti-inflammatory and antipyretic activities, the acetone extracts of leaves, at a dose of 400 mg/kg (p.o.) reduced significantly (p < 0.001) the pain, inflammation and fever responses in vivo. Bioactive compounds such as hyperin, chlorogenic acid, rutin and caffeic acids were identified in the leaves of P. leschenaultii employing HPLC–DAD–MS analysis. These findings illustrate the excellent potential of this species as valuable source of natural phytochemicals with pharmacological properties.     

Chemical composition and biological potential of seed oil and leaf extracts of Henophyton deserti Coss. & Durieu

Henophyton deserti was characterized in respect to its chemical composition, antioxidant potential and antimicrobial activity. Fourteen compounds were identified by LC/MS, GC/MS, and GC in leaf and seed extracts. Total flavonoids ranged between 45.66 and 181.2 mg QE g^?1 and 2.03 and 38.95 mg QE g^?1 dry weight (dw) in leaf and seed polar extracts, respectively. Rutin, Kaempferol 3-rutinoside, Diosmetin 7-O-Glucoside, and Acacetin 7-O-Glucoside flavonoids were tentatively identified in this plant. The profile of seed fatty acids revealed oleic acid (C18:1, 27%), linoleic acid (C18:2, 12%) and linolenic acid (C18:3, 17%). The highest antioxidant activities of 85.2% and 67.5% were obtained with methanol and ethyl acetate leaf extracts. All H. deserti extracts were active against at least one of the tested bacteria, leaf ethyl acetate extract showed the lowest MIC of 0.156 mg ml^?1. Only seed ethyl acetate extract showed antifungal activity with a MIC of 2.5 mg ml^?1.     

Anti-inflammatory and anti-oxidant activities of Mallotus oppositifolius (Geisel) methanol leaf extracts

The methanol leaf extract of Mallotus oppositifolius was evaluated for anti-inflammatory activity in rats and mice using acute and chronic anti-inflammatory models with acetylsalicylate acid (aspirin) as the reference drug. The antioxidant activity was done in vitro using ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-hydrazyl (DPPH) spectrophotometric assays. The extract dose dependently and significantly reduced paw edema volume in rats induced by carrageenan (p < 0.01), decreased croton oil-induced ear inflammation (p < 0.05), inhibited cotton pellet-induced granuloma in mice and reduced the rat paw thickness in formalin-induced arthritis.     

trans-Aconitic acid-based hetero-Diels-Alder reaction in the synthesis of thiopyrano[2,3-d][1,3]thiazole derivatives

The hetero-Diels-Alder reaction of 5-arylideneisorhodanines with trans-aconitic acid proceeds as a regio- and diastereoselective process with spontaneous decarboxylation of the [4+2]-adduct to furnish thiopyrano[2,3-d][1,3]thiazole (2) and chromeno[4′,3′:4,5]thiopyrano[2,3-d]thiazole (3) derivatives analogously to the use of itaconic acid as a dienophile. Conversely, the one-pot, three-component reaction of 5-arylideneisorhodanines, trans-aconitic acid and anilines proceeded without decarboxylation, leading to novel rel-(5′R,6′R,7′R)-5′-carboxy-7′-aryl-1-aryl-3′,7′-dihydro-2H,2′H,5H-spiro[pyrrolidin-3,6′-thiopyrano[2,3-d]thiazol]-2,2′,5-triones 4. Interestingly, the use of trans-aconitic acid trimethyl ester led to the opposite regioselectivity, yielding rel-(5R,6S,7S)-5-methyloxycarbonylmethyl-2-oxo-7-aryl-3,5,6,7-tetrahydro-2H-thiopyrano[2,3-d]thiazol-5,6-dicarboxylates 5. Selected compounds were examined for trypanocide activity against the bloodstream forms of Trypanosoma brucei where compound 4e showed the highest activity (IC₅₀ = 6.74 μM).     

Chemical composition and antioxidant, α-glucosidase inhibitory and antibacterial activities of three Echeveria DC. species from Mexico

Three Echeveria species from Sinaloa, Mexico (Echeveria craigiana, Echeveria kimnachii and Echeveria subrigida) were analyzed for their content of antioxidant compounds (β-carotene, ascorbic acid, α-tocopherol, total phenolics and flavonoids) and the in vitro antioxidant (DPPH, ABTS, ORAC and β-carotene bleaching [β-CBM]), α-glucosidase inhibitory and antibacterial activities. The studied Echeveria species showed high α-tocopherol content (2.9–9.0 mg/100 g f.w.) and total phenolics as Gallic Acid Equivalents (GAE) (152.2–400.5 mg GAE/100 g f.w.). Antioxidant activities of the three Echeveria methanol extracts (ME) were higher than those of other well-known plants with this property; the activities of E. craigiana (ABTS, 65.91 μmol ET/g f.w.) and E. subrigida (β-CBM, 79.3%) were remarkable. The Echeveria ME showed stronger α-glucosidase inhibition (IC₅₀ 25.21–50.57 μg/mL) than acarbose (IC₅₀ 3.59 mg/mL) as well as high antibacterial activity (Minimal Inhibitory Concentrations, MICs ⩽ 1 mg/mL), mainly against Gram positive bacteria. The results showed the three Echeveria species had components/biological activities with high potential for food/pharmacological uses and could be exploited by sustainable management schemes.     

Pseudoalteromone A: a novel bioactive ubiquinone from a marine bacterium Pseudoalteromonas sp. CGH2XX (Pseudoalteromonadaceae)

A novel ubiquinone derivative, pseudoalteromone A (1), possessing a 9C nor-monoterpenoid moiety was produced from a marine bacterium Pseudoalteromonas sp. CGH2XX. This bacterium is originally isolated from a cultured-type octocoral Lobophytum crassum. The structure of ubiquinone 1 was established by spectroscopic methods. Pseudoalteromone A (1) exhibited significant cytotoxicity toward the MOLT-4 (human acute lymphoblastic leukemia, IC₅₀ = 3.764 μg/mL) cells and displayed a significant inhibitory effect (inhibition rate 45.1%) on the release of elastase by human neutrophils at a concentration of 10 μg/mL.     

A novel visible spectrophotometric method for the determination of ethamsylate in pharmaceutical preparations and biological samples

A highly sensitive visible spectrophotometric method has been developed to determine ethamsylate in this paper, which is based on using Cu(II) as spectroscopic probe reagent. The study indicates that in the presence of SCN^? and KNO₃, Cu(II) is reduced to Cu(I) by ethamsylate at pH 5.0, and the in situ formed Cu(I) reacts with SCN^? to form into the white emulsion CuSCN that could be stayed upon the surface of water. According to the amount of residual Cu(II), the amount of ethamsylate can be indirectly determined. Under the optimal conditions, Beer's law is applicable in the range of 0.2–9.0 μg/mL (7.60 × 10^?7–3.42 × 10^?5 mol/L) for aqueous standard solution of ethamsylate with linear correlation coefficient of 0.9998. The detection limit and relative standard deviation are 0.12 μg/mL and 1.5%, respectively. And the molar absorption coefficient of the indirect determination of ethamsylate is 1.0 × 10⁵ L/mol cm. The method is successfully applied to determine ethamsylate in pharmaceutical preparations and biological samples.     

Gas Chromatography-Mass Spectroscopic,high performance liquid chromatographic and In-silico characterization of antimicrobial and antioxidant constituents of Rhus longipes (Engl)

Rhus longipes is one of those underutilized plant species with inherent values. Therefore, we conducted a phytochemical study and investigated the antioxidant and antimicrobial potentials of the plant extracts. Aqueous and ethanol extract was subjected to phytochemical analysis. Gas chromatography-mass spectroscopy was performed to identify the volatile compounds while high performance liquid chromatography was done to identify the phenolic and flavonoids in the ethanol extract. Bioactivity and molecular docking analysis was also done for the identification of the bioactive constituents. Tannins, flavonoids, phenolics, terpenoids, steroids, alkaloids, and saponins were identified both in the ethanol and in aqueous extracts of R. longipes. The extracts and ascorbic acid exhibited radical inhibition in a concentration dependent manner. The IC₅₀ values; 3.23, 4.13, 70.75 µg/mL (ABTS), 200.82, 103.63, 390.83 µg/mL (DPPH), 10.06, 93.46, 253.26 µg/mL (O₂), and 99.77, 109.23, 446.34 µg/mL (NO) for ascorbic acid, ethanol and aqueous extract respectively showed that ethanol extract exhibited better radical inhibition than the aqueous extract. 4-hydroxybenzoic and 4-hydroxycoumarin were the most abundant phenolics in the extract. The ethanol extract of R. longipes demonstrated broad-spectrum antibacterial activity with inhibition zone of 25.5 mm against S. aureus, 27.5 mm against E. coli, and 20.5 mm against P. aeruginosa. The identified phytochemicals demonstrated inhibitory potentials against bacterial glucosamine 6-phosphate synthase, penicillin-binding protein 3, DNA gyrase and β-lactamase. It is evident that R. longipes have some antioxidant and antibacterial properties and the plant contain important phytochemicals with ability to retard food oxidation and deterioration and thus could be annexed for various industrial and medicinal purposes.