Inhibitory effects of selected cannabinoids against dipeptidyl peptidase IV, an enzyme linked to type 2 diabetes.

Ethnopharmacological relevance: In recent times the decriminalisation of cannabis globally has increased its use as an alternative medication. Where it has been used in modern medicinal practises since the 1800s, there is limited scientific investigation to understand the biological activities of this plant. Aim of the study: Dipeptidyl peptidase IV (DPP-IV) plays a key role in regulating glucose homeostasis, and inhibition of this enzyme has been used as a therapeutic approach to treat type 2 diabetes. However, some of the synthetic inhibitors for this enzyme available on the market may cause undesirable side effects. Therefore, it is important to identify new inhibitors of DPP-IV and to understand their interaction with this enzyme. Methods: In this study, four cannabinoids (cannabidiol, cannabigerol, cannabinol and Δ9-tetrahydrocannabinol) were evaluated for their inhibitory effects against recombinant human DPP-IV and their potential inhibition mechanism was explored using both in vitro and in silico approaches. Results: All four cannabinoids resulted in a dose-dependent response with IC50 values of between 4.0 and 6.9 µg/mL. Kinetic analysis revealed a mixed mode of inhibition. CD spectra indicated that binding of cannabinoids results in structural and conformational changes in the secondary structure of the enzyme. These findings were supported by molecular docking studies which revealed best docking scores at both active and allosteric sites for all tested inhibitors. Furthermore, molecular dynamics simulations showed that cannabinoids formed a stable complex with DPP-IV protein via hydrogen bonds at an allosteric site, suggesting that cannabinoids act by either inducing conformational changes or blocking the active site of the enzyme. Conclusion: These results demonstrated that cannabinoids may modulate DPP-IV activity and thereby potentially assist in improving glycaemic regulation in type 2 diabetes.

Phytochemicals from Pterocarpus angolensis DC and Their Cytotoxic Activities against Breast Cancer Cells.

Pterocarpus anglonesis DC is an indigenous medicinal plant belonging to the Pterocarpus genus of the Fabaceae family. It is used to treat stomach problems, headaches, mouth ulcers, malaria, blackwater fever, gonorrhea, ringworm, diarrhea, heavy menstruation, and breast milk stimulation. Column chromatography of the stem bark extracts resulted in the isolation of eight compounds, which included friedelan-3-one (1), 3α-hydroxyfriedel-2-one (2), 3-hydroxyfriedel-3-en-2-one (3), lup-20(29)-en-3-ol (4), Stigmasta-5-22-dien-3-ol (5), 4-O-methylangolensis (6), (3ß)-3-acetoxyolean-12-en-28-oic acid (7), and tetradecyl (E)-ferulate (8). The structures were established based on NMR, IR, and MS spectroscopic analyses. Triple-negative breast cancer (HCC70), hormone receptor-positive breast cancer (MCF-7), and non-cancerous mammary epithelial cell lines (MCF-12A) were used to test the compounds' cytotoxicity. Overall, the compounds showed either no toxicity or very low toxicity to all three cell lines tested, except for the moderate toxicity displayed by lupeol (4) towards the non-cancerous MCF-12A cells, with an IC50 value of 36.60 µM. Compound (3ß)-3-acetoxyolean-12-en-28-oic acid (7) was more toxic towards hormone-responsive (MCF-7) breast cancer cells than either triple-negative breast cancer (HCC70) or non-cancerous breast epithelial (MCF-12A) cells (IC50 values of 83.06 vs. 146.80 and 143.00 µM, respectively).

Cytotoxicity and antimicrobial activity of isolated compounds from Monsonia angustifolia and Dodonaea angustifolia.

ETHNOPHARMACOLOGICAL RELEVANCE: Monsonia angustifolia is traditionally used to treat anthrax, heartburn, diarrhea, eye infections and hemorrhoids. Dodonaea angustifolia is frequently used as a treatment for dental pain, microbial infections and jungle fever. The two plant species were selected due to the presence of secondary metabolites such as coumarins, flavonoids, terpenoids, saponins and polyphenolics from the crude extracts, which exhibit pharmacological significance. The pure isolated compounds from the crude extracts are known for their diverse structures and interesting pharmacophores. AIM: To isolate and identify antibacterial and antifungal chemical constituents from Monsonia angustifolia and Dodonaea angustifolia plant extracts and evaluate the cytotoxicity of pure compounds from the crude extracts. MATERIALS AND METHODS: Extractives from M. angustifolia and D. angustifolia plants were isolated using chromatographic techniques and structures were elucidated based on NMR, IR and MS spectroscopic techniques. A microplate serial dilution method was used to evaluate the antibacterial activity of extracts and pure compounds against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and antifungal activity against Candida albicans and Cryptococcus neoformans. The cytotoxicity was determined using the 3-(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The dichloromethane, ethyl acetate and methanol crude extracts from the plants exhibited significant inhibition of microbial growth. The phytochemical investigation of these active crude extracts led to the isolation of five pure active compounds, 5-methoxyjusticidin A (1), cis-phytyl diterpenoidal fatty acid ester (2), stigmasterol (3), ß-sitosterol (4) and 5-hydroxy-7,4'-dimethoxyflavone (5). Stigmasterol (3) showed good antifungal activity against Cryptococcus neoformans with a minimum inhibition concentration (MIC) of 25 µg/mL and Candida albicans (MIC = 50 µg/mL). CONCLUSION: Compounds (1-5) isolated from Monsonia angustifolia and Dodonaea angustifolia showed antibacterial and antifungal activities and were non-toxic against Madin-Darby canine kidney (MDCK) cells and VERO monkey kidney (VERO) cells.

A Critical Review of the Antimicrobial and Antibiofilm Activities of Green-Synthesized Plant-Based Metallic Nanoparticles.

Metallic nanoparticles (MNPs) produced by green synthesis using plant extracts have attracted huge interest in the scientific community due to their excellent antibacterial, antifungal and antibiofilm activities. To evaluate these pharmacological properties, several methods or protocols have been successfully developed and implemented. Although these protocols were mostly inspired by the guidelines from national and international regulatory bodies, they suffer from a glaring absence of standardization of the experimental conditions. This situation leads to a lack of reproducibility and comparability of data from different study settings. To minimize these problems, guidelines for the antimicrobial and antibiofilm evaluation of MNPs should be developed by specialists in the field. Being aware of the immensity of the workload and the efforts required to achieve this, we set out to undertake a meticulous literature review of different experimental protocols and laboratory conditions used for the antimicrobial and antibiofilm evaluation of MNPs that could be used as a basis for future guidelines. This review also brings together all the discrepancies resulting from the different experimental designs and emphasizes their impact on the biological activities as well as their interpretation. Finally, the paper proposes a general overview that requires extensive experimental investigations to set the stage for the future development of effective antimicrobial MNPs using green synthesis.

In vitro antibacterial and cytotoxic effects of Euphorbia grandicornis Blanc chemical constituents.

BACKGROUND: Euphorbia grandicornis is widely utilized in traditional medicine for the treatment of microbial infections including sexually transmitted diseases such as syphilis, gonorrhoea and for healing of wounds. OBJECTIVE: The aim of this work was to isolate and evaluate the antibacterial and anticancer activities of Euphorbia grandicornis chemical constituents. METHODS: Chemical constituents were isolated and identified using various spectroscopic techniques such as IR, MS, and NMR. The single point growth inhibitory potential of the compounds was determined using a 96-well plate based assay. RESULTS: The CH2Cl2 crude extracts exhibited potent antibacterial activity against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538 with percentage growth of 94.90 ± 4.24 and 29.47 ± 4.89 respectively. Hence, the CH2Cl2 crude extract was further subjected to column chromatography which resulted in the isolation of methyl 2,5-dihydroxybenzoate (1), n-octyl benzoate (2), friedelanol (3), and germanicol (4) and identification of compounds 12-24 for the first time in the species based on the LC-MS/MS spectroscopic data. The purified compounds (1-4), and previously reported compounds (5-11) were evaluated for antibacterial activities against S. aureus and E. coli, as well as the cytotoxicity effects against HeLa cells. Of the purified compounds, methyl 2,5-dihydroxybenzoate (1), was the most active against E.coli and S. aureus with a percentage growth of 19.12 ± 0.65 and 23.32 ± 0.23 respectively. ß-amyrin (6), and ß-sitosterol (8), were active against S. aureus with percentage growth of 27.17 ± 0.07, and 47.79 ± 2.99 respectively. CONCLUSION: The results obtained from this study indicate that E. grandicornis, is a rich source of chemical constituents that may provide new lead compounds for the development of antibacterial agents.

Euphorbia Diterpenes: An Update of Isolation, Structure, Pharmacological Activities and Structure-Activity Relationship.

Euphorbia species have a rich history of ethnomedicinal use and ethnopharmacological applications in drug discovery. This is due to the presence of a wide range of diterpenes exhibiting great structural diversity and pharmacological activities. As a result, Euphorbia diterpenes have remained the focus of drug discovery investigations from natural products. The current review documents over 350 diterpenes, isolated from Euphorbia species, their structures, classification, biosynthetic pathways, and their structure-activity relationships for the period covering 2013-2020. Among the isolated diterpenes, over 20 skeletal structures were identified. Lathyrane, jatrophane, ingenane, ingenol, and ingol were identified as the major diterpenes in most Euphorbia species. Most of the isolated diterpenes were evaluated for their cytotoxicity activities, multidrug resistance abilities, and inhibitory activities in vitro, and reported good activities with significant half-inhibitory concentration (IC50) values ranging from 10-50 µM. The lathyranes, isopimaranes, and jatrophanes diterpenes were further found to show potent inhibition of P-glycoprotein, which is known to confer drug resistance abilities in cells leading to decreased cytotoxic effects. Structure-activity relationship (SAR) studies revealed the significance of a free hydroxyl group at position C-3 in enhancing the anticancer and anti-inflammatory activities and the negative effect it has in position C-2. Esterification of this functionality, in selected diterpenes, was found to enhance these activities. Thus, Euphorbia diterpenes offer a valuable source of lead compounds that could be investigated further as potential candidates for drug discovery.

Review of the Traditional Uses, Phytochemistry, and Pharmacological Activities of Rhoicissus Species (Vitaceae).

Species within the genus Rhoicissus (Vitaceae) are commonly used in South African traditional medicine. The current review discusses the occurrence, distribution, traditional uses, phytochemistry, and pharmacological properties of Rhoicissus species covering the period 1981-2020. The data reported were systematically collected, read, and analysed from scientific electronic databases including Scopus, Scifinder, Pubmed, and Google Scholar. Reported evidence indicates that species in this genus are used for the treatment of gastrointestinal complaints, sexually transmitted infections (STIs), and infertility, as well as to tone the uterus during pregnancy and to facilitate delivery. Pharmacological studies have further shown that members of the Rhoicissus genus display antidiabetic, uterotonic, ascaricidal, hepatoprotective, antioxidant, antimicrobial, anticancer, and anti-inflammatory properties. They are linked to the presence of bioactive compounds isolated from the genus. Hence, Rhoicissus species can potentially be an alternative therapeutic strategy to treat diseases and develop safer and more potent drugs to combat diseases. Plant species of this genus have valuable medicinal benefits due to their significant pharmacological potential. However, scientific investigation and information of the therapeutic potential of Rhoicissus remain limited as most of the species in the genus have not been fully exploited. Therefore, there is a need for further investigations to exploit the therapeutic potential of the genus Rhoicissus. Future studies should evaluate the phytochemical, pharmacological, and toxicological activities, as well as the mode of action, of Rhoicissus crude extracts and secondary compounds isolated from the species.

Green synthesis of antimicrobial silver nanoparticles using aqueous leaf extracts from three Congolese plant species (Brillantaisia patula, Crossopteryx febrifuga and Senna siamea).

In the present study, silver nanoparticles (AgNPs) were synthesized using aqueous leaf extracts of three Congolese plant species, namely Brillantaisia patula (BR-PA), Crossopteryx febrifuga (CR-FE) and Senna siamea (SE-SI). The obtained AgNPs were studied for their optical, structural, surface morphological and antibacterial properties. The prepared AgNPs were characterized by using UV-Visible spectra, Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray spectroscopy (EDX) and X-ray diffractometer (XRD). The synthesized nanoparticles were spherical shaped and well-dispersed with average sizes ranging from 45 to 110 nm. The AgNPs derived from BR-PA, CR-FE and SE-SI exhibited higher antibacterial activity against three bacterial pathogens of the human skin compared to their respective crude extracts and AgNO3. This indicated that the biomolecules covering the nanoparticles may enhance the biological activity of metal nanoparticles. Hence, our results support that biogenic synthesis of AgNPs from Congolese plants constitutes a potential area of interest for the therapeutic management of microbial diseases such as infectious skin diseases.

Antiparasitic Constituents of Beilschmiedia louisii and Beilschmiedia obscura and Some Semisynthetic Derivatives (Lauraceae).

The MeOH/CH2Cl2 (1:1) extracts of the roots and leaves of Beilschmiedia louisii and B. obscura showed potent antitrypanosomal activity during preliminary screening on Trypanosoma brucei brucei. Phytochemical investigation of these extracts led to the isolation of a mixture of two new endiandric acid derivatives beilschmiedol B (1) and beilschmiedol C (2), and one new phenylalkene obscurene A (3) together with twelve known compounds (4-15). In addition, four new derivatives (11a-11d) were synthesized from compound 11. Their structures were elucidated based on their NMR and MS data. Compounds 5, 6, and 7 were isolated for the first time from the Beilschmiedia genus. Additionally, the NMR data of compound 4 are given here for the first time. The isolates were evaluated for their antitrypanosomal and antimalarial activities against Tb brucei and the Plasmodium falciparum chloroquine-resistant strain Pf3D7 in vitro, respectively. From the tested compounds, the mixture of new compounds 1 and 2 exhibited the most potent antitrypanosomal activity in vitro with IC50 value of 4.91 µM.

Cordidepsine is A Potential New Anti-HIV Depsidone from Cordia millenii, Baker.

Chemical investigation of Cordia millenii, Baker resulted in the isolation of a new depsidone, cordidepsine (1), along with twelve known compounds including cyclooctasulfur (2), lup-20(29)-en-3-triacontanoate (3), 1-(26-hydroxyhexacosanoyl)glycerol (4), glyceryl-1-hexacosanoate (5) betulinic acid (6), lupenone (7), ß-amyrone (8), lupeol (9), ß-amyrin (10), allantoin (11), 2'-(4-hydroxyphenyl)ethylpropanoate (12) and stigmasterol glycoside (13). Hemi-synthetic reactions were carried out on two isolated compounds (5 and 6) to afford two new derivatives, that is, cordicerol A (14) and cordicerol B (15), respectively. The chemical structures of all the compounds were established based on analysis and interpretation of spectroscopic data such as electron ionization mass spectrometry (EI-MS), high resolution electrospray ionization mass spectrometry (HR-ESI-MS), fast atom bombardment mass spectrometry (FAB-MS), one dimension and two dimension nuclear magnetic resonance (1D and 2D-NMR) spectral data as well as X-ray crystallography (XRC). Lupeol ester derivatives [Lup-20(29)-en-3-triacontanoate (3)], monoglycerol derivatives [1-(26-hydroxyhexacosanoyl)glycerol (4) and glyceryl-1 hexacosanoate (5)] were isolated for the first time from Cordia genus while sulfur allotrope [cyclooctasulfur (2)] was isolated for the first time from plant origin. Biological assays cordidepsine (1) exhibited significant anti-HIV integrase activity with IC50 = 4.65 µM; EtOAc extract of stem barks, EtOAc fraction of roots and leaves were not toxic against 3T3 cells.

Phytochemical, anti-inflammatory and anti-trypanosomal properties of Anthocleista vogelii Planch (Loganiaceae) stem bark.

ETHNOPHARMACOLOGICAL RELEVANCE: Anthocleista vogelii Planch (Loganiaceae) is used in African Traditional Medicine for the treatment of pain and inflammatory disorders as well as sleeping sickness. AIM OF THE STUDY: To determine the in vivo anti-inflammatory and in vitro anti-trypanosomal activities of the extracts of A. vogelii stem bark and identify the phytochemical classes of the fractions responsible for the activities. MATERIALS AND METHODS: The in vivo anti-inflammatory activity of the extracts was evaluated using the egg albumin-induced rat paw oedema model while the in vitro anti-trypanosomal activity was assessed on Trypanosoma brucei brucei. The in vitro cytotoxicity was assessed on HeLa (human cervix adenocarcinoma) cell line. RESULTS: The methanolic extract of A. vogelii stem bark, with 11.2% yield, gave LD50 > 5000 mg/kg. The n-hexane fraction of the extract contains steroids, terpenes and fatty acids and yielded non-cytotoxic terpenoidal column fraction with anti-trypanosomal IC50 of 3.0 µg/mL. The ethylacetate fraction at 100 mg/kg dose significantly (p < 0.05) provoked 37.8, 62.5 and 69.7% inhibition of oedema induced by egg-albumin at the second, fourth and sixth hours respectively. CONCLUSION: The study demonstrated that the anti-inflammatory and anti-trypanosomal activities of A. vogelii are probably due to non-cytotoxic terpenoids and validated the traditional use of A. vogelii in the treatment of inflammation and sleeping sickness.

Anti-HIV-1 integrase potency of methylgallate from Alchornea cordifolia using in vitro and in silico approaches.

According to the 2018 report of the United Nations Programme on HIV/AIDS (UNAIDS), acquired immune deficiency syndrome (AIDS), a disease caused by the human immunodeficiency virus (HIV), remains a significant public health problem. The non-existence of a cure or effective vaccine for the disease and the associated emergence of resistant viral strains imply an urgent need for the discovery of novel anti-HIV drug candidates. The current study aimed to identify potential anti-retroviral compounds from Alchornea cordifolia. Bioactive compounds were identified using several chromatographic and spectroscopic techniques and subsequently evaluated for cytotoxicity and anti-HIV properties. Molecular modelling studies against HIV-1 integrase (HIV-1 IN) were performed to decipher the mode of action of methylgallate, the most potent compound (IC50 = 3.7 nM) and its analogues from ZINC database. Cytotoxicity assays showed that neither the isolated compounds nor the crude methanolic extract displayed cytotoxicity effects on the HeLa cell line. A strong correlation between the in vitro and in silico results was observed and important HIV-1 IN residues interacting with the different compounds were identified. These current results indicate that methylgallate is the main anti-HIV-1 compound in A. cordifolia stem bark, and could be a potential platform for the development of new HIV-1 IN inhibitors.

Synthesis of silver nanoparticles from a Desmodium adscendens extract and its antibacterial evaluation on wound dressing material.

The one-pot synthesis of silver nanoparticles (AgNPs) using the medium-polar extract of Desmodium adscendens (Sw.) DC. is presented here as an alternative synthesis of metal NPs. Characterisation of the formed NPs showed polydispersed AgNPs ranging from 15 to 100 nm where the concentration of metal ions was found to play a role in the size and shape of the prepared NPs. It could be established that the flavonoids, saponins, and alkaloids present in the extract acted as both reducing and stabilising agents during the formation of the capped metal NPs. This means of NP synthesis was also employed during the in situ immobilisation of AgNPs on gauze and plaster. An evaluation of the antibacterial activity of the medium-polar D. adscendens extract, AgNPs suspended in solution, and the immobilised AgNPs against Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778), and Escherichia coli (ATCC 25922) showed high efficacy against the latter in particular. This suggests that gauze, dilute silver nitrate solutions, and D. adscendens extract could be used successfully in the simple in situ preparation of effective antibacterial wound dressings.

Anti-malarial synergy of secondary metabolites from Morinda lucida Benth.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots, stem and leaves of Morinda lucida are used in some African countries as treatment against different types of fevers including yellow fever, malaria, trypanosomiasis and feverish conditions during child birth. AIM OF THE STUDY: To determine the in vitro cell toxicity and anti-malarial activity of the extracts of stem bark of M. lucida and to identify the secondary metabolites in the extract that may be responsible for this activity. MATERIALS AND METHODS: The cell toxicity studies of crude extract [dichloromethane (DCM): Methanol (MeOH) in a ratio of1:1 (v/v)] as well as compounds isolated from the same extract were carried out using human cervix adenocarcinoma cells (HeLa cells); while the anti-malarial activities of the same samples were performed against Plasmodium falciparum strain 3D7 using the parasite lactate dehydrogenase (pLDH) assay. The isolation of the active compounds was carried out using chromatographic techniques (column and thin layer chromatography) where as mass spectrometry (MS), Fourier transform infrared spectroscopy (FTIR) as well as 1D- and 2D- nuclear magnetic resonance (NMR) analyses were employed in the characterisation and identification of the isolated secondary metabolites. RESULTS: The pLDH and cell toxicity assays for the crude extract and the fractions of M. lucida indicated that some fractions reduced the malaria parasite viability by approximately 50% at 100µg/mL and they were not significantly cytotoxic. An IC50 done on the crude extract gave a value of 25µg/mL. The % cell viability for the crude extract in cell toxicity assay remained at 100%. Seven chemical constituents i.e. asperuloside (1), asperulosidic acid (2), stigmasterol (3a), ß-sitosterol (3b), cycloartenol (3c), campesterol (3d) and 5,15-O-dimethylmorindol (4) were isolated from the DCM-MeOH extract of stem bark. The isolated compounds tested were not that active by themselves individually at 20µM but their activities were increased when the isolated compounds were combined. As seen when compounds 2, 3 and 4 (% viability: 93, 123 and 101 respectively) were combined yielding an IC50 value of 17µM. Furthermore, this is the first report of compounds 1, 2, 3c, 3d and 4 isolated from M. lucida. CONCLUSION: The crude extract completely suppressed the growth of P. falciparum. This indicates that the crude extract contains many compounds that might be acting in synergy. The observed activity of the crude extract and the samples containing a mixture of different compounds support the traditional use of M. lucida for the treatment of malaria.

Cytotoxicity, phytochemical analysis and antioxidant activity of crude extracts from rhizomes of Elephantorrhiza elephantina and Pentanisia prunelloides.

BACKGROUND: Elephantorrhiza elephantina (Ee) and Pentanisia prunelloides (Pp) are two medicinal plants which are widely used to remedy various ailments including diarrhoea, dysentery, inflammation, fever, rheumatism, heartburn, tuberculosis, haemorrhoids, skin diseases, perforated peptic ulcers and sore joints in southern Africa (South Africa, Swaziland, Botswana and Zimbabwe). The following study was conducted to explore the in vitro cytotoxicity, antioxidant properties and phytochemical profile of the two medicinal plants. MATERIALS AND METHODS: The cytotoxicity of the aqueous and methanol extracts and fractions of both species was studied using the brine shrimp lethality tests (BST) for the first time. RESULTS: The results demonstrated that the lethality (LC50) for crude extracts for both plants ranged between 1.8 and 5.8 ppm and was relatively greater than that for the methanol, ethyl acetate and chloroform fractions of the extracts which ranged between 2.1 ppm and 27 ppm. This suggested that crude extracts were more potent than their respective fractions, further explaining that the different fractions of phytochemicals in these plant species work jointly (in synergy) to exert their therapeutic efficacy. Both aqueous and methanol extracts of the two medicinal plants demonstrated a high degree of antioxidant capacity against the DPPH radical with the Duh and Yen inhibition percentage ranging between 4.5% and 72%. Phytochemical studies of the rhizome extracts showed that the major compounds present include flavonoids, tannins, anthocyanidins, anthraquinones, triterpenoids (oleanolic acid), the steroidal saponin Diosgenin, the sugars, rhamnose, glucuronic acid, Arabinose and hexoses. CONCLUSION: This is the first report of the detection and isolation of diosgenin and oleanolic acid from the rhizome extracts of Ee and Pp. All structures were determined using spectroscopic/spectrometric techniques (1H NMR and 13C and LC-ESI-MS) and by comparison with literature data.

Determination of catechins from Elephantorrhiza elephantina and Pentanisia prunelloides using voltammetry and UV spectroscopy.

In this work, we report the identification and quantification of catechins by electrochemistry and UV-Vis spectroscopy in Elephantorrhiza elephantina (Fabaceae) and Pentanisia prunelloides (Rubiaceae), both of which are medicinal plants that are widely used in Southern Africa to remedy various ailments. A comparative study of the catechin content as (-)-epicatechin equivalent is reported for the first time, with E. elephantina exhibiting a higher concentration relative to P. prunelloides in both aqueous and methanol extracts.