Ethyl linoleate

Influence of Sucrose and Activated Charcoal on Phytochemistry and Vegetative Growth in Zephyranthes irwiniana (Ravenna) Nic. Garcia (Amaryllidaceae).[Pubmed:38475416]

Plants (Basel). 2024 Feb 20;13(5):569.

Zephyranthes irwiniana (Ravenna) Nic. Garcia is an endemic, red list threatened species from the Brazilian savanna (Cerrado) with pharmacological potential to treat the symptoms of Alzheimer's Disease (AD). This work analyzed the vegetative growth and phytochemistry of its potential compounds, in response to variations in sucrose concentration and activated carbon (AC). Seeds were germinated in vitro and in the greenhouse. The in vitro bulbs were separated in six treatments with different sucrose concentrations (30, 45 and 60 gL(-1)) and/or AC (1 gL(-1)). Biomass increases in individuals grown in the greenhouse were higher than those cultivated in vitro. Sucrose concentration significantly increased biomass and root number. AC had a positive influence on leaf and root size, and a negative influence on root number. GC-MS analyses indicated great variation in the abundance of alpha-terpenyl-acetate, Ethyl linoleate, clionasterol and lycorine between treatments, with maximum concentrations of 53.06%, 38.68, 14.34% and 2.57%, respectively. Histolocalization tests indicated the presence of alkaloids in the leaf chlorenchyma and bulb cataphylls. Finally, the present study provided new evidence that the constitution of the culture medium directly influences the vegetative growth and phytochemistry of this species, providing a good medium condition for propagating the species under threat.

[A new benzopyran glycoside from Gentiana macrophylla].[Pubmed:38403317]

Zhongguo Zhong Yao Za Zhi. 2024 Jan;49(2):412-419.

Thirteen compounds were isolated and identified from 70% ethanol extract of the roots of Gentiana macrophylla by multi-chromatographic methods, including microporous resin, silica gel, and C_(18) reversed-phase column chromatography, as well as HPLC as follows: macrophylloside G(1), macrophylloside D(2), 5-formyl-2,3-dihydroisocoumarin(3),(+)-medicarpin(4),(+)-syringaresinol(5), liquiritigenin(6),(3R)-sativanone(7),(3R)-3'-O-methylviolanone(8), 4,2',4'-trihydroxychalcone(9), latifolin(10), gentioxepine(11), 6alpha-hydroxycyclonerolidol(12), and Ethyl linoleate(13). Compound 1 was a new benzopyran glycoside. Compounds 4, 6-10, 12, and 13 were isolated for the first time from Gentiana plants. Compounds 1 and 2 showed promising hepatoprotective activity against D-GalN-induced AML12 cell damage at the concentration of 10 mumol.L~(-1), and compound 2 exhibited more significant activity than silybin at the same concentration.

Elucidation of the mechanism of Zhenbao pills for the treatment of spinal cord injury by network pharmacology and molecular docking: A review.[Pubmed:38363936]

Medicine (Baltimore). 2024 Feb 16;103(7):e36970.

To explore the mechanism of the Zhenbao pill (ZBP) in treating spinal cord injury (SCI). The TCMSP Database, HERB Database and literature search were used to screen the effective ingredients and targets of ZBP; SCI-related genes were searched in GeneCards, OMIM, PharmGkb, TTD and DrugBank databases; the potential targets of ZBP for treating SCI were predicted and Venn diagrams were drawn, and the "herb-ingredient-target" network was constructed by Cytoscape software. The PPI network was constructed by STRING software, and the core targets were screened by cytoNCA plug-in; GO enrichment and KEGG pathway analysis were performed on the predicted targets using the DAVID Platform, and visualized with the Microbiology Network Platform. The molecular docking between the key ingredients and the core target was carried out by AutoDockVina software. 391 active ingredients and 836 action targets were obtained from ZBP and there are 1557 SCI related genes in 5 disease databases. The top 5 active ingredients were Quercetin, Camptothecin, Kaempferol, Ethyl iso-allocholate, and Ethyl linoleate, and 5 core genes were SRC, CTNNB1, TP53, AKT1, and STAT3. GO enrichment analysis showed that the core targets were involved in 1206 biological processes, 120 cellular components and 160 molecular functions; KEGG enrichment analysis showed that the core targets involved 183 pathways, including PI3K-Akt signaling pathway and other signaling pathways. Molecular docking indicated that CTNNB1, SRC, TP53, AKT1 and STAT3 showed good binding ability with the active ingredients quercetin, kaempferol and ethyl isobutyric acid. ZBP improves SCI through multi-components, multi-targets and multi-pathways.

Solvent-free synthesis of hydrophobic and amphiphilic esters using a chemically modified lipase from Thermomyces lanuginosus: a comparative study with native and immobilized forms.[Pubmed:38169079]

Chembiochem. 2024 Feb 16;25(4):e202300843.

Using lipases to catalyze the synthesis of the most differentiated type of compounds remains one of the major challenges among scientists. Seeking more economic and advantageous catalysts is a current goal of green chemistry. In this work, we demonstrate the potential of a chemically modified form of lipase from Thermomyces lanuginosus (cmLTL) for the synthesis of both hydrophobic (heptyl heptanoate, heptyl octanoate, heptyl decanoate, decyl heptanoate, decyl octanoate and decyl decanoate) and amphiphilic (2-(2-ethoxyethoxy)ethyl oleate and 2-(2-ethoxyethoxy)Ethyl linoleate) esters, in bulk. The results were compared with its native (LTL) and immobilized (imLTL) forms. The data revealed that LTL showed poor activity for all reactions performed with n-heptane (eta<20 %). ImLTL was able to synthesize all hydrophobic esters (eta>60 %), with exception of the short ester, heptyl heptanoate. cmLTL was the only form of LTL capable of producing hydrophobic and amphiphilic esters, without compromising the yield when the reactions were performed under solvent-free conditions (>50 %). Molecular modeling showed that the active pocket of cmLTL is able to deeply internalize transcutol, with stronger interactions, justifying the outstanding results obtained. Furthermore, owing to the possibility of cmLTL filtration, the reusability of the catalyst is ensured for at least 6 cycles, without compromising the reaction yields.

Study of the Chemical Composition of Rosa beggeriana Schrenk's Fruits and Leaves.[Pubmed:37765460]

Plants (Basel). 2023 Sep 18;12(18):3297.

Rosa species are widely used in folk medicine in different countries of Asia and Europe, but not all species are studied in-depth. For instance, Rosa beggeriana Schrenk, a plant which grows in Central Asia, Iran, and some parts of China, is little described in articles. Column and thin-layer chromatography methods were used to isolate biologically active substances. From a study of fruits and leaves of Rosa beggeriana Schrenk, a large number of compounds were identified, seven of which were isolated: 3beta,23-dihydroxyurs-12-ene (1), beta-sitosterol (2), betulin (3), (+)-catechin (4), lupeol (5), Ethyl linoleate (6), and ethyl linolenoate (7). Their structures were elucidated by (1)H, DEPT and (13)C NMR spectroscopy, mass spectrometry, and GC-MS (gas chromatography-mass spectrometry). The study also identified the structures of organic compounds, including volatile esters and acids. Consequently, comprehensive data were acquired concerning the chemical constitution of said botanical specimen.

Insecticidal potential of a Consolida ajacis extract and its major compound (ethyl linoleate) against the diamondback moth, Plutella xylostella.[Pubmed:37666618]

Pestic Biochem Physiol. 2023 Sep;195:105557.

The diamondback moth (Plutella xylostella) is one of the most destructive lepidopteran pests of cruciferous vegetables. However, DBM has developed resistance to current chemical and biological insecticides used for its control, indicating the necessity for finding new insecticides against it. Bio-insecticides derived from plant extracts are eco-friendly alternatives to synthetic pesticides. The aims of this study were to evaluate the insecticidal activity of Consolida ajacis seed extracts against DBM, the underlying mechanism of the control effect of promising extracts, and the identification of the main insecticidal compounds of these extracts. The results showed that ethyl acetate extract of C. ajacis seed exhibited strong contact toxicity (LC(50): 5.05 mg/mL), ingestion toxicity, antifeedant, and oviposition deterrent activities against DBM, among the extracts evaluated. At 72 h, glutathiase, acetylcholinesterase, carboxylesterase, peroxidase, and superoxide dismutase activities were inhibited, but catalase activity was activated. The main compound identified from the extract was Ethyl linoleate, which had the most significant insecticidal activity on the diamondback moths. This study's findings provide a better understanding of the insecticidal activity of ethyl acetate extract obtained from C. ajacis and its main component (Ethyl linoleate). This will help in the development of new insecticides to control DBM.

Metagenomic Insights into the Regulatory Effects of Microbial Community on the Formation of Biogenic Amines and Volatile Flavor Components during the Brewing of Hongqu Rice Wine.[Pubmed:37628073]

Foods. 2023 Aug 16;12(16):3075.

As one of the typical representatives of Chinese rice wine (Huangjiu), Hongqu rice wine is produced with glutinous rice as the main raw material and Hongqu as the fermentation starter. The complex microbial flora in the brewing process may have a great influence on the formation of the flavor quality and drinking safety of Hongqu rice wine. Previous studies have shown that high biogenic amine (BA) content in rice wine has potential physiological toxicity and has become a bottleneck problem restricting the development of the rice wine industry. This study aimed to evaluate the regulatory effects of the microbial community on the formation of BAs and volatile flavor components during the brewing of Hongqu rice wine. The results demonstrated that histamine, putrescine, cadaverine, tyramine, tryptamine, spermine, and spermidine were the main BAs in Hongqu rice wine. The contents of putrescine, cadaverine, histamine, tyramine, and spermidine in Hongqu rice wine of HBAs (with higher BAs content) were significantly higher than those in LBAs (with lower BAs content). GC-MS testing results showed that there were significant differences in the composition of volatile organic compounds (VOCs) between HBAs and LBAs. Among them, VOCs such as 2-methoxy-4-vinylphenol, ethyl caprate, phenethyl acetate, ethyl lactate, ethyl myristate, ethyl palmitate, ethyl n-octadecanoate, ethyl oleate, and Ethyl linoleate were identified as the characteristic volatile components with significant differences between HBAs and LBAs. Microbiome analysis based on metagenomic sequencing revealed that unclassified_g_Pantoea, Klebsiella pneumoniae, Panobacter disperse, unclassified_f_Enterobacteriaceae, Leuconostoc mesenteroides, and Saccharomyces cerevisiae were the dominant microbial species in the HBA brewing process, while Weissella confuse, Pediococcus acidilactici, Saccharomyces cerevisiae, and Aspergillus niger were the dominant microbial species in the LBA brewing process. Furthermore, correlation heatmap analysis demonstrated that BAs were positively related to Lactobacillus curvatus, Lactococcus lactis, and Leuconostoc mesenteroides. Bioinformatical analysis based on the KEGG database revealed that the microbial genes encoding enzymes involved in BAs' synthesis were more abundant in HBAs, and the abundances of microbial genes encoding enzymes related to BAs' degradation and the metabolism of characteristic volatile components were higher in LBAs. Overall, this work provides important scientific data for enhancing the flavor quality of Hongqu rice wine and lays a solid foundation for the healthy development of the Hongqu rice wine industry.

Linoleic acid from the endophytic fungus Diaporthe sp. HT-79 inhibits the growth of Xanthomonas citri subsp. citri by destructing the cell membrane and producing reactive oxygen species (ROS).[Pubmed:37105613]

Pestic Biochem Physiol. 2023 May;192:105423.

Citrus canker disease caused by Xanthomonas citri subsp. citri (Xac) severely influences the quality and quantity of citrus fruits. The current management of this disease mainly relies on the application of copper-associated chemicals, which poses a threat to human health and the environment. The present study isolated an endophytic fungus HT-79 from the healthy navel orange tree, whose crude fermentation product significantly inhibited the growth of Xac. The strain HT-79 was identified as a species of the Diaporthe genus. The petroleum ether extract (PEE) of the crude fermentation product of HT-79 exhibited remarkable activity against Xac with a MIC (minimum inhibitory concentration) value of 0.0625 mg/mL, significantly better than the positive control CuSO(4) (MIC = 0.125 mg/mL). Bioassay-guided isolation of PEE resulted in the discovery of one highly potent anti-Xac subfraction, namely fraction 5 (MIC = 0.0156 mg/mL). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that fraction 5 mainly consisted of palmitic acid (18.17%), ethyl palmitate (15.66%), linoleic acid (6.80%), oleic acid (18.32%), Ethyl linoleate (21.58%), ethyl oleate (15.87%), and ethyl stearate (3.60%). Among these seven compounds, linoleic acid (MIC = 0.0078 mg/mL) was found to be the most potent against Xac, followed by oleic acid (MIC = 0.0156 mg/mL), while all others were less pronounced than CuSO(4). Linoleic acid highly inhibited the growth of Xac via the destruction of the cell membrane and overproduction of reactive oxygen species (ROS). A preliminary in vivo experiment revealed that linoleic acid was effective in the control of citrus canker disease.

[Determination of four fatty acid ethyl esters in olive oil by solid phase extraction-gas chromatography].[Pubmed:37005923]

Se Pu. 2023 Apr;41(4):359-365.

The fatty acid ethyl ester (FAEE) content of olive oil is an important indicator of its quality. At present, the international standard method used to detect FAEEs in olive oil is silica gel (Si) column chromatography-gas chromatography (GC); however, this technique presents a number of disadvantages, including complex operation, long analysis times, and high reagent consumption. In this study, a method based on Si solid phase extraction (SPE)-GC was established to determine four FAEEs in olive oil, namely, ethyl palmitate, Ethyl linoleate, ethyl oleate, and ethyl stearate. First, the effects of the carrier gas were investigated, and He gas was ultimately selected as the carrier gas. Next, several internal standards were screened, and ethyl heptadecenoate (cis-10) was determined as the optimal internal standard. The SPE conditions were also optimized, and the effects of different brands of Si SPE columns on the recoveries of analytes were compared. Finally, a pretreatment method in which 0.05 g of olive oil was extracted with n-hexane and purified through a Si SPE column (1 g/6 mL) was developed. A sample could be processed within approximately 2 h using a total reagent volume of about 23 mL. Validation of the optimized method revealed that the four FAEEs have good linearities within the range of 0.1-5.0 mg/L (coefficients of determination (R(2))>0.999). The limits of detection (LODs) of the method were within 0.78-1.11 mg/kg, and its limits of quantification (LOQs) were in the range of 2.35-3.33 mg/kg. The recoveries ranged from 93.8% to 104.0% at all spiked levels tested (4, 8, and 20 mg/kg), and the relative standard deviations were 2.2%-7.6%. Fifteen olive oil samples were tested using the established method, and the total FAEEs of three extra-virgin olive oil samples were found to exceed 35 mg/kg. Compared with the international standard method, the proposed method has the advantages of simpler pretreatment process, shorter operation time, lower reagent consumption and detection cost, high precision, and good accuracy. The findings provide an effective theoretical and practical reference for improving olive oil detection standards.

Bakuchiol and Ethyl (Linoleate/Oleate) Synergistically Modulate Endocannabinoid Tone in Keratinocytes and Repress Inflammatory Pathway mRNAs.[Pubmed:36992949]

JID Innov. 2022 Dec 26;3(3):100178.

The endocannabinoid (eCB) system plays an active role in epidermal homeostasis. Phytocannabinoids such as cannabidiol modulate this system but also act through eCB-independent mechanisms. This study evaluated the effects of cannabidiol, bakuchiol (BAK), and ethyl (linoleate/oleate) (ELN) in keratinocytes and reconstituted human epidermis. Molecular docking simulations showed that each compound binds the active site of the eCB carrier FABP5. However, BAK and Ethyl linoleate bound this site with the highest affinity when combined 1:1 (w/w), and in vitro assays showed that BAK + ELN most effectively inhibited FABP5 and fatty acid amide hydrolase. In TNF-stimulated keratinocytes, BAK + ELN reversed TNF-induced expression shifts and uniquely downregulated type I IFN genes and PTGS2 (COX2). BAK + ELN also repressed expression of genes linked to keratinocyte differentiation but upregulated those associated with proliferation. Finally, BAK + ELN inhibited cortisol secretion in reconstituted human epidermis skin (not observed with cannabidiol). These results support a model in which BAK and ELN synergistically interact to inhibit eCB degradation, favoring eCB mobilization and inhibition of downstream inflammatory mediators (e.g., TNF, COX-2, type I IFN). A topical combination of these ingredients may thus enhance cutaneous eCB tone or potentiate other modulators, suggesting novel ways to modulate the eCB system for innovative skincare product development.

Pro-Apoptotic and Anti-Cancer Activity of the Vernonanthura Nudiflora Hydroethanolic Extract.[Pubmed:36900417]

Cancers (Basel). 2023 Mar 6;15(5):1627.

The mitochondrial voltage-dependent anion channel 1 (VDAC1) protein is involved in several essential cancer hallmarks, including energy and metabolism reprogramming and apoptotic cell death evasion. In this study, we demonstrated the ability of hydroethanolic extracts from three different plants, Vernonanthura nudiflora (Vern), Baccharis trimera (Bac), and Plantago major (Pla), to induce cell death. We focused on the most active Vern extract. We demonstrated that it activates multiple pathways that lead to impaired cell energy and metabolism homeostasis, elevated ROS production, increased intracellular Ca(2+), and mitochondria-mediated apoptosis. The massive cell death generated by this plant extract's active compounds involves the induction of VDAC1 overexpression and oligomerization and, thereby, apoptosis. Gas chromatography of the hydroethanolic plant extract identified dozens of compounds, including phytol and Ethyl linoleate, with the former producing similar effects as the Vern hydroethanolic extract but at 10-fold higher concentrations than those found in the extract. In a xenograft glioblastoma mouse model, both the Vern extract and phytol strongly inhibited tumor growth and cell proliferation and induced massive tumor cell death, including of cancer stem cells, inhibiting angiogenesis and modulating the tumor microenvironment. Taken together, the multiple effects of Vern extract make it a promising potential cancer therapeutic.

Molecular Screening of Bioactive Compounds of Garlic for Therapeutic Effects against COVID-19.[Pubmed:36831179]

Biomedicines. 2023 Feb 20;11(2):643.

An outbreak of pneumonia occurred on December 2019 in Wuhan, China, which caused a serious public health emergency by spreading around the globe. Globally, natural products are being focused on more than synthetic ones. So, keeping that in view, the current study was conducted to discover potential antiviral compounds from Allium sativum. Twenty-five phytocompounds of this plant were selected from the literature and databases including 3-(Allylsulphinyl)-L-alanine, Allicin, Diallyl sulfide, Diallyl disulfide, Diallyl trisulfide, Glutathione, L-Cysteine, S-allyl-mercapto-glutathione, Quercetin, Myricetin, Thiocysteine, Gamma-glutamyl-Lcysteine, Gamma-glutamylallyl-cysteine, Fructan, Lauricacid, Linoleicacid, Allixin, Ajoene, Diazinon Kaempferol, Levamisole, Caffeicacid, Ethyl linoleate, Scutellarein, and S-allylcysteine methyl-ester. Virtual screening of these selected ligands was carried out against drug target 3CL protease by CB-dock. Pharmacokinetic and pharmacodynamic properties defined the final destiny of compounds as drug or non-drug molecules. The best five compounds screened were Allicin, Diallyl Sulfide, Diallyl Disulfide, Diallyl Trisulfide, Ajoene, and Levamisole, which showed themselves as hit compounds. Further refining by screening filters represented Levamisole as a lead compound. All the interaction visualization analysis studies were performed using the PyMol molecular visualization tool and LigPlot+. Conclusively, Levamisole was screened as a likely antiviral compound which might be a drug candidate to treat SARS-CoV-2 in the future. Nevertheless, further research needs to be carried out to study their potential medicinal use.

Efficacy and safety of Politranexamide(R) liposomal emulsion on facial melasma: A comparative study.[Pubmed:36718844]

J Cosmet Dermatol. 2023 Jun;22(6):1780-1785.

INTRODUCTION: Melasma is a human melanogenesis dysfunction that results in localized, chronic acquired hypermelanosis of the skin difficult to treat. METHODS: This prospective, randomized, single-blind, study aimed to compare the efficacy and tolerability of a liposomal emulsion based on Politranexamide(R) (SAMPLE A) with that of a competitor based on acetylglucosamine, Ethyl linoleate and phenyl ethyl resorcinol (SAMPLE B) in patients affected by facial melasma on 26 patients. Disease severity was assessed by the Melasma Area Severity Index (MASI) at baseline and after 6 and 12 weeks of therapy. All patients were subjected to photo documentation using DermaView camera and Antera 3D camera. RESULTS: The mean MASI score at baseline was 10.93 +/- 7 in the group A and 9.34 +/- 6.29 in the group B, respectively. A significant decrease in MASI score from baseline was noted in both treatment groups as early as 6 weeks of follow-up (p = 0.00096 for SAMPLE A and p = 0.0049 for SAMPLE B) and was confirmed at the end of the treatment (p = 0.0006 for SAMPLE A and p = 0.00039 for SAMPLE B). Intergroup comparison revealed a greater improvement of melasma among patients in group A compared to those in group B that was quite statistically significant at weeks 6 (p = 0.055009) and significant after 12 weeks of follow-up (p = 0.032942). Both treatment groups experienced an improvement in Antera average level of melanin. CONCLUSION: Our results suggested Politranexamide(R) to be a useful and safe therapeutic option in treating melasma, more effective than competitor used in this study.

The palliative effect of mulberry leaf and olive leaf ethanolic extracts on hepatic CYP2E1 and caspase-3 immunoexpression and oxidative damage induced by paracetamol in male rats.[Pubmed:36637651]

Environ Sci Pollut Res Int. 2023 Mar;30(14):41682-41699.

This study investigated the possible protective role of mulberry leaf (MLE) and olive leaf (OLE) ethanolic extracts against paracetamol (PTL)-induced liver injury in rats compared to silymarin as a reference drug. Initially, MLE and OLE were characterized using gas chromatography-mass spectrometry (GC/MS). Then, forty male Sprague Dawley rats were divided into five groups: the negative control group orally received distilled water for 35 days, the PTL-treated group (PTG) received 500 mg PTL/kg b. wt. for 7 days, the MLE-treated group (MLTG) received 400 mg MLE/kg b. wt., the OLE-treated group (OLTG) received 400 mg OLE/kg b. wt., and the silymarin-treated group (STG) received 100 mg silymarin/kg b. wt. The last three groups received the treatment for 28 days, then PTL for 7 days. The GC-MS characterization revealed that MLE comprised 19 constituents dominated by Ethyl linoleate, phytol, hexadecanoic acid, ethyl ester, and squalene. Moreover, OLE comprised 30 components, and the major components were 11-eicosenoic acid, oleic acid, phytol, and a-tetralone. MLE and OLE significantly corrected the PTL-induced normocytic normochromic anemia, leukocytosis, hypercholesterolemia, and hypoproteinemia. Moreover, the MLE and OLE pretreatment considerably suppressed the PTL-induced increment in serum levels of hepatic enzymes, including alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase. Furthermore, the PTL-induced depletion in antioxidant enzymes, including glutathione peroxidase, superoxide dismutase, and catalase, and the rise in hepatic malondialdehyde content were significantly reversed by the MLE and OLE pretreatment. Besides, MLE and OLE pretreatment significantly protected the hepatic tissue against PTL-induced DNA damage, pathological perturbations, and increased caspase 3 and CYP2E1 immunoexpression. Of note, OLTG showed better enhancement of most indices rather than MLTG. Conclusively, these findings imply that OLE, with its antioxidant and antiapoptotic capabilities, is superior to MLE in protecting against PTL-induced liver injury.

Activation of Vegetable Oils by Reaction with Maleic Anhydride as a Renewable Source in Chemical Processes: New Experimental and Computational NMR Evidence.[Pubmed:36500234]

Molecules. 2022 Nov 23;27(23):8142.

Vegetable oils are bio-based and sustainable starting materials that can be used to develop chemicals for industrial processes. In this study, the functionalization of three vegetable oils (grape, hemp, and linseed) with maleic anhydride was carried out either by conventional heating or microwave activation to obtain products that, after further reactions, can enhance the water dispersion of oils for industrial applications. To identify the most abundant derivatives formed, trans-3-octene, methyl oleate, and Ethyl linoleate were reacted as reference systems. A detailed NMR study, supported by computational evidence, allowed for the identification of the species formed in the reaction of trans-3-octene with maleic anhydride. The signals in the (1)H NMR spectra of the alkenyl succinic anhydride (ASA) moieties bound to the organic chains were clearly identified. The reactions achieved by conventional heating were carried out for 5 h at 200 degrees C, resulting in similar or lower amounts of ASA units/g of oil with respect to the reactions performed by microwave activation, which, however, induced a higher viscosity of the samples.