The extraction of effective components and an antioxidant activity study of Tulipa edulis

Plant extracts from natural sources are an excellent choice for food additives and natural antioxidants.In this study,the active components of Tulipa edulis were extracted and analysed,and their antioxidant capacity was measured.Then,the crude extract mixture was separated and purified using a Sephadex LH-20 gel,and the antioxidant activity of the purified products was determined.Human umbilical vein endothelial human umbilical vein endothelial cells(HUVEC)cells were treated with 35 mmol/L glucose to construct a model of oxidative stress.Then,the cells were treated with the active component to observe whether the products of T.edulis could have a good protective effect on HUVEC cells induced by glucose.Transcriptome analysis was also performed on HUVEC cells after same treatment to explore the possible mechanism of the component F2 protecting HUVEC cells from oxidative stress induced by high glucose.The results showed that component F2 obtained from T.edulis has strong antioxidant activity.Moreover,F2 can play a strong antioxidant protective role in HUVEC cells.Meanwhile,the gene expression of heme oxygenase 1(HO-1),γ-glutamyl cysteine ligase catalytic subunit(GCLC)and NAD(P)H quinone oxidoreductase-1(NQO1)in HUVEC cells was up-regulated after treated with F2.This study provides reference value for the further development and application of T.edulis and the d evelopment of functional food.

Fatty Acid Composition of Hazelnut Kernel Oil from Coula edulis Collected in the Republic of Congo

Coula edulis is non-timber forest product (NTFP) used in Africa for its hazelnuts, which contain edible seeds with a demonstrated nutritional potential. However, there have been very few scientific studies of this species in the Republic of Congo. Thus, the aim of the current study was therefore to determine the fatty acid composition of the oil extracted from Coula edulis hazelnut seeds collected at random in the Republic of Congo. The oil was extracted using the Soxhlet method and its fatty acid composition was determined by gas chromatography. The extracted oil from Coula edulis hazelnut kernels is rich in monounsaturated fatty acids (95.28%), particularly oleic acid (94.5%), which classifies it as an oleic oil and gives it interesting nutritional and therapeutic properties. On the other hand, saturated fatty acids (4.15%) and polyunsaturated fatty acids (0.35%) are not well represented. Its low poly-unsaturated fatty acid content makes it more stable when stored at room temperature.

Studies of the Variability of Primary Metabolites in the Epicarp, Mesocarp and Seed of Dacryodes edulis Fruit at Taste Maturity

The fruits of Dacryodes edulis are rich in biologically active substances, which makes them of great interest in terms of validation. In this study, we targeted the primary metabolites in the epicarp, the mesocarp and the seed of the fruit of Dacryodes edulis at taste maturity, which were selected for their nutritional quality and its appreciation throughout the Gulf of Guinea area, which is very popular because of its large size, texture and special taste. The evaluation of total carbohydrates, total lipids and soluble proteins in the epicarp, mesocarp and seed of the fruit at taste maturity was made from spectrophotometer measurements. The overall analysis of the results of the present study shows that total carbohydrates, total lipids and proteins accumulate more in the seed with respectively 251.33 ± 1.15 mg/g DM;9.92 ± 0.201 mg/g DM and 55.075 ± 0.024 mg/g DM. Likewise, the results indicate low concentrations of total carbohydrates and total lipids in the epicarp with respectively 245 ± 1 mg/g DM and 4.77 ± 0.047 mg/g DM, on the other hand, it is the mesocarp which presents the lowest content of soluble proteins: 28.075 ± 3.231 mg/g DM. This variation could be linked to the nature of the compartment, more particularly to the storage location. This comparative study could lead to the valorization of the seed of the fruit of Dacryodes edulis for its richness in metabolites and arouse significant interest in nutrition.

Effects of Ethanol Condensation Reflux Extraction and Ultrasound-assisted Extraction on the Content of Total Flavonoids of Pueraria edulis

With Pueraria edulis from different habitats in Yunnan Province,Guangdong Province and Guangxi Zhuang autonomous region as materials,the medicinal materials of P.edulis were extracted by ethanol condensation reflux extraction and ultrasound-assisted extraction,and the effects of different extraction methods the contents of total flavonoids in P.edulis from different areas were compared.The results showed that the best extraction time for P.edulis in Yunnan Province was 20 min,and that for Guangdong and Guangxi Zhuang Autonomous Region was 30 min.The yield of total flavonoids from P.edulis extracted by the condensation reflux extraction method was relatively better.The content of total flavonoids in P.edulis in Yunnan was the highest with an average content of 0.2557%,while the average content of total flavonoids in P.edulis in Guangdong was the lowest with an average content of 0.2108%.

Morphological beak differences ofloliginid squid, Uroteuthis chinensis and Uroteuthis edulis, in the northern South China Sea

The mitre squid(U roteuthis chinensis) and the swordtip squid( U. edulis) are Indo-Pacific cephalopod species that are abundant in the western Pacific Ocean. They are currently exploited in the East and South China Seas and make up a significant portion of the Chinese neritic squid catch. Beaks, the feeding organs of squid, are important for individual size and biomass estimation because of their high resistance to degradation in predator stomachs and consistent dimensions. In this study, 104 U. chinensis and 143 U. edulis individuals were sampled from northern South China Sea with mantle length from 70 to 260 mm and 96 to 284 mm, respectively. The results indicated that morphological beak values were greater for U. edulis, compared to U. chinensis, for upper hood length(UHL), upper crest length(UCL), upper lateral wall length(ULWL), lower crest length(LCL), and lower lateral wall length(LLWL). According to principal component analysis, UHL/ML, UCL/ML, ULWL/ML, LCL/ML, LLWL/ML and LWL/ML could represent the characteristics of beaks for U. chinensis, while UHL/ML, UCL/ML, ULWL/ML, LHL/ML, LCL/ML and LLWL/ML could represent it for U. edulis. According to Akaike's information criterion(AIC) values, a power function was the most suitable model for U. chinensis, while a linear function was the most suitable model for U. edulis. The beak variable-mantle length ratio(beak variable/mantle length) declined with the increasing of mantle length and declined sharply at the early stage of growth in both beaks and species. The ratio changed quickly after achieving the mantle length of 140 mm for U. chinensis, while the ratio changed quickly after 170 mm for U. edulis. Beaks in both species experienced sharper changes through maturity stage I to II than other maturity stages. This study gives us basic beak morphology information for U. chinensis and U. edulis in the East and South China Seas. Geometric morphological methods combined with dietary analysis should be used in the future.

Factors affecting soil organic carbon in a Phyllostachys edulis forest

Phyllostachys edulis plays an important role in maintaining carbon cycling.We examined the effects of soil properties on organic carbon content in a P.edulis forest on Dagang Mountain,Jiangxi Province,China.Based on correlation and stepwise multiple regression analyses,the effects of seven soil factors on organic carbon and their sensitivities to change were studied using path and sensitivity analyses.The results revealed differences in the interconnections and intensities of soil factors on organic carbon.Soil porosity,field capacity,and ammonium nitrogen levels were the main factors affecting organic carbon in the ecosystem.Soil porosity had a strong direct effect on organic carbon content and a strong indirect effect through field capacity.Field capacity and ammonium nitrogen levels mainly affected organic carbon directly.Field capacity,soil porosity,and ammonium nitrogen content,as well as bulk density,b-glucosidase activity,and invertase activity,were sensitive factors.Polyphenol oxidase activity was insensitive.Our study provides a theoretical basis for understanding the effects of soil factors on organic carbon,which can be utilised to improve P.edulis forest management strategies and promote carbon sequestration capacities.

Effects of Carpobrotus edulis Extract on Oxidative Stress and 158N Oligodendrocyte Death

Objective Age-related diseases, including neurodegenerative diseases, are associated with oxidative stress and lipid peroxidation, and increase the levels of cholesterol auto-oxidation products such as 7β-hydroxycholesterol(7β-OHC). Thus, it is imperative to identify agents that can prevent 7β-OHC-induced side-effects. Methods We evaluated the potential protective effects of Carpobrotus edulis ethanol-water extract(EWe) on murine oligodendrocytes(158 N) cultured in the absence or presence of 7β-OHC(20 μg/mL, 24 h). The cells were incubated with EWe(20-200 μg/mL) 2 h before 7β-OHC treatment. Mitochondrial activity and cell growth were evaluated with the MTT assay. Photometric methods were used to analyze antioxidant enzyme [catalase(CAT) and glutathione peroxidase(GPx)] activities and the generation of lipid and protein oxidation products [malondialdehyde(MDA), conjugated diene(CD), and carbonylated proteins(CPs)]. Results Treatment with 7β-OHC induced cell death and oxidative stress(reflected by alteration in CAT and SOD activities). Overproduction of lipid peroxidation products(MDA and CDs) and CPs was also reported. The cytotoxic effects associated with 7β-OHC were attenuated by 160 μg/mL of EWe of C. edulis. Cell death induced by 7β-OHC treatment was ameliorated, GPx and CAT activities were restored to normal, and MDA, CD, and CP levels were reduced following C. edulis extract treatment. Conclusion These data demonstrate the protective activities of C. edulis EWe against 7β-OHC-induced disequilibrium in the redox status of 158 N cells, indicative of the potential role of this plant extract in the prevention of neurodegenerative diseases.

Relationship between oxygen concentration, respiration and filtration rate in blue mussel Mytilus edulis

The large water-pumping and particle-capturing gills of the filter-feeding blue mussel Mytilus edulis are oversized for respiratory purposes. Consequently, the oxygen uptake rate of the mussel has been suggested to be rather insensitive to decreasing oxygen concentrations in the ambient water, since the diff usion rate of oxygen from water flowing through the mussel determines oxygen uptake. We tested this hypothesis by measuring the oxygen uptake in mussels exposed to various oxygen concentrations. These concentrations were established via N2-bubbling of the water in a respiration chamber with mussels fed algal cells to stimulate fully opening of the valves. It was found that mussels exposed to oxygen concentrations decreasing from 9 to 2 mg O_2/L resulted in a slow but significant reduction in the respiration rate, while the filtration rate remained high and constant. Thus, a decrease of oxygen concentration by 78% only resulted in a 25% decrease in respiration rate. However, at oxygen concentrations below 2 mg O_2/L M. edulis responded by gradually closing its valves, resulting in a rapid decrease of filtration rate, concurrent with a rapid reduction of respiration rate. These observations indicated that M.edulis is no longer able to maintain its normal aerobic metabolism at oxygen concentration below 2 mg O 2/L, and there seems to be an energy-saving mechanism in bivalve molluscs to strongly reduce their activity when exposed to low oxygen conditions.

Comparison of Fatty Acid and Proximate Composition between <i>Holothuria edulis</i>and <i>Holothuria scabra</i>Collected from Coastal Water of Sabah, Malaysia

The nutritional values of different species of sea cucumber are greatly concerned because of their dietary and curative properties. In this study, two species of sea cucumber, Holothuria edulis, a low-valued noncommercial species, and Holothuria scabra, a high-valued commercial species were selected to compare its proximate composition and fatty acids. H. edulis a prevalent species in coastal water of Sabah is not commercially importance like H. scabra. Sea cucumbers were captured live from the Sabah marine habitat. All samples were immediately eviscerated, freeze-dried and stored at 4°C in until analyzed. Silylating agent N, O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) was used to derivitization of fatty acid prior to gas chromatography-mass spectrometry (GC-MS) analysis. Proximate compositions (%), such as moisture, crude protein, crude lipids and ash were carried using standard methods. Major fatty acids in H. edulis and H. scabra were saturated fatty acid (SFA) accounted for 83.95% and 98.60%, respectively and dominated with Palmitic acids. Polyunsaturated fatty acid (PUFA), arachidonic acid of 16.05% was found only in H. edulis, but absent H. scabra. Proximate compositions (dry weight) were varied greatly within these two species. Moisture, crude protein, crude lipids and ash of 85.5%, 70.5%, 1.37% and 1.27% respectively were obtained in H. edulis. On the other hand 84.5% of moisture, 51.2% of crude protein, 0.27% of crude lipids and 4.44% of ashes were determined in H. scabra. Significantly higher protein (p H. edulis compare to H. scabra could be the choice of option for the utilization of this non commercial species as nutraceutical industry and also alternatives to reduce the pressure on heavily exploited species of H. scabra.

Relationships between daily growth of different groups of swordtip squid(Uroteuthis edulis)and environmental variables in the East China Sea

Swordtip squid(Uroteuthis edulis)is one of the important economical fishing target species in the East China Sea.Uroteuthis edulis is characterized by rapid growth,extensive migration,and long spawning period and sensitive to surrounding environment.In order to assess its stock status,it is necessary to explore its spawning season,growth patterns of different populations and their relationship with the environment in advance.In this paper,based on the samples of U.edulis collected in the East China Sea from September 2017 to March 2018,we explored the relationships between daily growth of statolith microstructure and environmental variables by gradient forest method and generalized additive model.The spawning season of U.edulis was found to be nearly one year,and two dominant season groups were found:the spring group with the peak period of April and the summer group with the peak period of August.Water temperature in the depth of 25 m(Temp_25),sea surface temperature(SST)and zonal velocity were the key environmental variables for the daily growth of April-spawning group.The most suitable environmental conditions for the growth of April-spawning group were water temperature(24-27℃)and velocity(0.1-0.3 m/s).SST,Temp_25 and mixed layer depth were the key environment variables for the daily growth of August-spawning group.The most suitable environmental conditions for the growth of Augustspawning group were water temperature(21-28℃)and water depth(0-50 m).Key environmental variables of different groups suggested that early growth was significantly affected by seasonal changes of water temperature,current velocity and prey abundance.This study explored the relationships between early growth and environmental variables and provided the scientific guidance for the management and conservation of U.edulis.

Dynamic Changes of Chemical and Mechanical Properties of Moso Bamboo (Phyllostachys edulis) Culms under Different Storage Conditions

The durability of bamboo based on its chemical and mechanical properties is a crucial consideration for the wood-based industry due to its vulnerability to insects and microorganisms.We investigated the dynamic changes of chemical and mechanical properties of Phyllostachys edulis under air-dry and water storage conditions for 3,6 and 12 months respectively.The chemical properties of P.edulis bamboo culms varied with culm age but insignificantly with culm height.The mechanical properties of P.edulis culms showed an increasing trend with culm age.Water storage condition decreased the ash,SiO2 and lignin content,but increased the ethanol-benzene extracts.It also created an anaerobic environment for bamboo culms in which only anaerobic respiration was possible contributing to reducing the content of soluble sugar and starch,thereby beneficial for decreasing the damage from insects and microorganisms.Moreover,the water storage conditions could maintain culm mechanical performance better.Therefore,the indigenous practice of local people to store bamboo culms in ponds has good science behind it and water storage practices of bamboo culms was recommended.

Non-isoprenoid botryane sesquiterpenoids from basidiomycete Boletus edulis and their cytotoxic activity

Three non-isoprenoid botryane sesquiterpenoids,named boledulins A-C(1-3),have been isolated from the cultures of basidiomycete Boletus edulis Bull.The structures were established by means of spectroscopic methods.Boledulin A(1)exhibited moderate inhibitory activity against five human cancer cell lines.

紫贻贝(Mytilus edulis Linnaeus)大量死亡原因的初步研究

从发病紫贻贝(Mytilusedulis)的内脏团内分离出一株菌SS1,通过人工感染试验,证实其为致病菌。经形态、生理生化等几项指标鉴定,该菌株性状与溶藻弧菌基本相符。药物敏感试验结果表明羧苄青霉、万古霉素、克林霉素、氨苄青霉素、阿莫西林等9种药物对该菌株均有明显的抑制作用。

Bio-Fabrication of Silver Nanoparticles Using Catha edulis Extract: Procedure Optimization and Antimicrobial Efficacy Encountering Antibiotic-Resistant Pathogens

The current study aimed to optimize a green synthesis procedure for fabricating biogenic silver nanoparticles (AgNPs) using an aqueous extract of Catha edulis (Qat;khat) leaves. The final product was characterized using various analytical techniques. Parameters’ optimization including pH, contact time, temperature, and amount of leaf extract were carried out. AgNPs formation was confirmed by UV-vis spectra at 403 nm, FT-IR, and XRD peaks. FTIR spectra showed the presence of various biochemical metabolites which played a critical role in the bio-reduction, capping, and stabilization of AgNPs. The biogenic AgNPs were spherical in shape with an average size between 27 and 32 nm as estimated from XRD and SEM images. Biogenic AgNPs showed significant activities against sensitive and multi-drug resistance Escherichia coli and Staphylococcus aureus strains. In addition, the experimental results proved that AgNPs have higher efficacy than antifungal drugs that are commonly used to treat Candida albicans oral infections.

Effect of Salinity on Growth of Mussels,Mytilus edulis,with Special Reference to Great Belt(Denmark)

The effects of salinities between 10 and 30 psu on the growth of blue mussels, Mytilus edulis, were studied in laboratory feeding experiments and compared to the growth of mussels suspended in net-bags in the brackish water Great Belt, Denmark. In the laboratory, 3 series of growth experiments were conducted: in Series #1, groups of mussels were exposed to 10, 15, 25 and 30 psu, in Series #2, two groups of mussels were exposed to 10 and 30 psu, respectively, for 15 days (first period) where upon the mussels were exposed to the reversed salinities for another 15 days (second period). In Series #3, two groups of mussels were initially exposed to 15 and 25 psu for 22 days whereupon the mussel groups were exposed to the reversed salinities for another 17 days. In the laboratory experiments there was a tendency towards reduced growth with decreasing salinity, reflected as reduced shell growth rate and decreasing weight specific growth rate with falling salinity. The shell growth rate was relatively low in the first feeding period compared to the second period, and mussels that were initially exposed to 10 psu, where the growth was low, exhibited fast growth when subsequently exposed to 30 psu, and reversed when 30 psu mussels were exposed to 10 psu. The study showed that mussels are able to adjust growth at changing salinities, and the observed effect of salinity could partly be explained by a temporary shell valve closure after a sudden change in salinity. The specific growth rate of mussels measured in laboratory experiments at salinities between 15 to 25 psu (4.2% to 4.8% d–1) were comparable to the growth of mussels in the field experiment (3.2% to 4.0% d–1) where the salinity varied between 24 and 13 psu during the growth period.

Changes in foliar nitrogen resorption of Phyllostachys edulis with culm development

Leaf nitrogen resorption is very important to Phyllostachys edulis development because the withdrawn nitrogen can help newly emerging and growing culms.However, few studies have focused on the ontogenetic changes in leaf nitrogen resorption of P. edulis. Here, we examined the variability in mature leaf nitrogen concentrations(Nm), nitrogen resorption efficiency(NRE) and proficiency(NRP or Ns) and leaf-level nitrogen use efficiency(NUE) of the current-, 3 rd-and 5 th-year culms in P.edulis stands under extensive management. Analyses of variance and correlation indicated that patterns of Nm,NRP, NRE and NUE were markedly affected by culm age and leaf nitrogen status. Nm, Nsand NRE were significant higher in younger(current-year) culms with 1-year lifespan leaves, while NUE was markedly higher in older(3 rd-or 5 th-year) culms with 2-year lifespan leaves. Significant linear correlations between Nmand NRP, NRE and NUE,Nmand NUE, Nsand NRE were found for each culm age,and Nmwas significantly positively correlated to NRE for all culms pooled. Higher proficiency in older culms led to higher NUE and lower NRE, these relationships can be modulated by Nm, which in turn, is restrained by leaf N availability and acquisition. Our results revealed that at the intraspecific level, P. edulis can adjust its leaf NRE, NRP,and leaf-level NUE in concert with culm development.Understanding nitrogen resorption characteristics and NUE of P. edulis can help decision-makers design appropriate deforestation strategies and achieve precise N fertilization for sustainable bamboo forest management.

Characterization of Extracts from the Bark of the Gabon Hazel Tree (Coula edulis baill) for Antioxidant,Antifungal and Anti-termite Products

Chemical composition of the bark extracts of Coula edulis was investigated to find potential antioxidant,anti-termite and antifungal compounds which can find useful applications in the fields of food,nutraceuticals,cosmetics or agrochemical.Phytochemical screening revealed the presence of several groups of active molecules such as alkaloids,polyphenols,flavonoids,saponins and sterols and/or terpenes in the different extracts.Total phenols,condensed tannins and flavonoids contents corroborated phytochemical screening.Gas chromatographymass spectrometry(GC-MS)analysis revealed compounds in dichloromethane extract different from those obtained with all the other solvents.Hexadecanoic and trans-9-octadecenoic acids,as well as stigmasterol andβ-sitosterol have been identified as the major compounds in the dichloromethane extract.Extracts obtained with acetone and toluene/ethanol mixture(2/1,v/v)indicated the presence of few amounts of fatty acids and sugars,catechin in small amount and huge amounts of phenolic acids like gallic and ellagic acids.The radical 2,2-diphenyl-1-picrylhydrazyle(DPPH)and the cationic radical 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS+)were used for evaluation of antioxidant properties of the different extracts.The dichloromethane extracts had a very low antioxidant activity,while acetone and toluene/ethanol extracts presented EC50 values similar to those of catechin and BHT used as reference antioxidant compounds.Effect of the different extracts of the bark of C.edulis on fungal growth inhibition indicated better inhibition of the mycelium growth of brown rot fungi compared to white rot fungi.Low anti-termite activities were recorded with the aqueous extracts,while stronger activities were recorded with dichloromethane,acetone and toluene/ethanol extracts.

Screening of Optimal Differentiation Medium to Lonicera edulis

An excellent Lonicera edulis strain, L1-8 that was bred by Northeast Institute of Geography and Agro-ecology, Chinese Academy of Sciences, was used as material in this research. The axillary buds of its dormant branches were used as explants. A fourfactor and four-level orthogonal test was designed in order to choose the best differentiation medium for providing the technical support of Lonicera edulis micropropagation. The results showed that the culture medium and concentration of 6-BA were the main factors, and the best differentiation condition was MS culture medium containing 2.0 mg · L-1 6-BA, 0.3 mg · L-1 IBA and 1.5 mg · L-1 GA3.

Beneficial effects of high-pressure homogenization on the dispersion stability of aqueous hydrolysate from Mytilus edulis

Owing to the formation of aggregation and gelation during storage,certain proteins and peptides exhibit limited applications in aqueous protein food products.The purpose of this study was to investigate the influence of homogenization and xanthan gum addition on the dispersion stability of Mytilus edulis hydrolysate(PHM).High-pressure homogenization(HPH)at 360 and 40 bar in the first and second values,respectively,and adding xanthan gum at a concentration of 1 mg/mL showed significantly improvement on the stability of the PHM solution.PHM-xanthan gum solutions(PHMX)showed the highest polypeptide precipitation rate and turbidity retention rate compared with those of PHM.Moreover,the centrifugal precipitation rate of PHMX without HPH was higher than that of homogeneous PHMX.After HPH treatment at 400 bar,the percentage of smaller particles in PHM and PHMX was increased,the aqueous system became more uniform,and the fluorescence intensity reached its maximum.HPH pretreatment improved the polypeptide dispersion stability and turbidity retention rate of PHM and PHMX and reduced the fluorescence intensity.The interactions of xanthan gum and polypeptide render the network microstructure more uniform under the conditions of homogenization,thus improving the dispersion stability of PHMX solutions.Therefore,under the premise of adding xanthan gum,HPH can better enhance the dispersion stability of the polypeptide in PHM.

Effects of Rising Temperature on Secondary Compounds of Yeheb (<i>Cordeauxia edulis</i>Hemsley)

The effects of temperature on net photosynthesis and stomatal conductance, emission of foliar volatile organic compounds (VOCs), and phenolics were investigated after exposing Cordeauxia edulis seedlings to control (27/19°C) and three levels of elevated (32/23, 37/27, or 42/31°C) day/night temperature regimes in controlled growth chambers. Emissions of foliar VOC were measured on 7th and 14th day (d) of exposures, whereas net photosynthesis and stomatal conductance were measured on the 8th and 15th d. Net photosynthesis and stomatal conductance were not significantly affected by elevated temperatures. Emission rate of isoprene increased by 4-fold with 10°C rise from the control on 7th d of exposure. Emission rates of monoterpenes, sesquiterpenes and total isoprenoids increased to 2-5-fold higher than that of control plants with 5°C rise. Foliar isoprene emission peaked at daytime maximum of 37°C and the mono- and sesquiterpenes at 32°C. Few individual foliar phenolics, and total foliar phenolics showed significant concentration differences between treatments. Although high VOC emissions under warming appeared to help plants to sustain abiotic stresses, arid/semi-arid species might substantially release highly reactive compounds that affect atmospheric chemistry. Hence, more studies are required on plant species of arid/semi-arid ecosystems of Africa to estimate the emission patterns and their role in atmospheric chemistry under the predicted future atmospheric warming.