Artepillin C,a Major Ingredient of Brazilian Propolis,Induces a Pungent Taste by Activating TRPA1 Channels

Brazilian green propolis is a popular health supplement because of its various biological properties. The ethanol extract of Brazilian green propolis (EEBP) is characteristic for its herb-like smell and unique pungent taste. However, the ingredients responsible for its pungency have not yet been identified. This study provides the first evidence that artepillin C is the main pungent ingredient in EEBP and that it potently activates human transient receptor potential ankyrin 1 (TRPA1) channels. EEBP was fractionated using column chromatography with a step gradient elution of an ethanol-water solution, and the fractions having the pungent taste were determined by sensory tests. HPLC analysis revealed that the pungent fraction was composed primarily of artepillin C, a prenylated derivative of cinnamic acid. Artepillin C was also identified as the pungent compound of EEBP by organoleptic examiners. Furthermore, the effects of artepillin C and other cinnamic acids found in EEBP on TRPA1 channels were examined by calcium imaging and plate reader-based assays in human TRPA1-expressing cells to investigate the molecular mechanisms underlying their pungent tastes. Artepillin C and baccharin activated the TRPA1 channel strongly, whereas drupanin caused a slight activation and p-coumaric acid showed no activation. Because the EC₅₀ values of artepillin C, baccharin, and allyl isothiocyanate were 1.8 µM, 15.5 µM, and 6.2 µM, respectively, artepillin C was more potent than the typical TRPA1 agonist allyl isothiocyanate. These findings strongly indicate that artepillin C is the main pungent ingredient in EEBP and stimulates a pungent taste by activating TRPA1 channels.     

Chemical composition and antibacterial activity of Cordia verbenacea extracts obtained by different methods

The present study describes the chemical composition and the antibacterial activity of extracts from Cordia verbenacea DC (Borraginaceae), a traditional medicinal plant that grows widely along the southeastern coast of Brazil. The extracts were obtained using different extraction techniques: high-pressure operations and low-pressure methods. The high-pressure technique was applied to obtain C. verbenacea extracts using pure CO₂ and CO₂ with co-solvent at pressures up to 30 MPa and temperatures of 30, 40 and 50 °C. Organic solvents such as n-hexane, ethyl acetate, ethanol, acetone and dichloromethane were used to obtain extracts by low-pressure processes. The antibacterial activity of the extracts was also subjected to screening against four strains of bacteria using the agar dilution method. The extraction yields were up to 5.0% w/w and up to 8.6% w/w for supercritical fluid extraction with pure CO₂ and with ethyl acetate as co-solvent, respectively, while the low-pressure extraction indicates yields up to 24.0% w/w in the soxhlet extraction using water and aqueous mixture with 50% ethanol as solvents. The inhibitory activity of the extracts in Gram-positive bacteria was significantly higher than in Gram-negative. The quantification and the identification of the extracts recovered were accomplished using GC/MS analysis. The most important components identified in the extract were artemetin, β-sitosterol, α-humulene and β-caryophyllene, among others.     

Inhibitory activity of Brazilian green propolis components and their derivatives on the release of cys-leukotrienes

The effects of Brazilian green propolis ethanol extract on Cry j1-induced cys-leukotrienes and histamine release from peripheral leukocytes of patients with allergic rhinitis were investigated. One of the key mechanisms for the anti-allergic properties of the extract was revealed to be the suppression of cys-LTs release. Furthermore, a series of propolis components and their phenethyl esters were synthesized and evaluated as inhibitors of cys-LTs release. Artepillin C, baccharin, and kaempferide were the major active components of the ethanol extract. The inhibitory activity of artepillin C phenethyl ester was comparable to that of existing LT synthesis inhibitors.     

Phenolic Acid Composition,Antiatherogenic and Anticancer Potential of Honeys Derived from Various Regions in Greece

The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content.     

Development of a protocol for supercritical carbon dioxide extraction of ubiquinone-10 from dried biomass of <Emphasis Type="Italic">Pseudomonas diminuta</Emphasis>

Ubiquinone (Coenzyme Q10, CoQ10), a yellow-to-orange-colored lipophilic substance having nutraceutical value, was extracted from dried biomass of Pseudomonas diminuta using supercritical carbon dioxide (SC-CO₂). The effect of different operational parameters (temperature, pressure, and extraction time) and addition of co-solvent on SC-CO₂ extraction of CoQ10 was studied in detail. The solubility parameter of CoQ10, CO₂, and CO₂ with ethanol and methanol as co-solvents was calculated and validated with experimental results. Theoretically, ethanol and methanol had significant effect as co-solvent, and the difference between the two was only marginal. A maximum recovery of 22.33% was obtained using pure SC-CO₂ at 40 °C, 150 bar, and run time of 60 min. Ethanol as co-solvent at 3 mL/g of dried biomass at 40 °C and 150 bar increased the recovery from 22.33 to 68.57%. Further optimization of the extraction conditions by Box–Behnken design effectively increased the recovery to 96.2%. The optimized conditions were a temperature of 38 °C, pressure of 215 bar, and run time of 58 min.     

Supercritical carbon dioxide extraction of marigold at high pressures: comparison of analytical and pilot-scale extraction

The effects of pressure and co-solvent on the extraction of anti-inflammatory faradiol esters in marigold (Calendula officinalis L.) were investigated by supercritical fluid extraction at laboratory and pilot scales. Pressures higher than 300 bar and modifier (ethanol) concentrations ranging from 0 to 20% (v/v) were used at an extraction temperature of 50 degrees C. With an analytical extractor, exhaustive extraction of the drug and highest concentrations in the extracts were achieved with 0.5% ethanol at the maximum pressure of 689 bar. Increased modifier concentrations improved the extractability at lower pressure, but the higher amount of total extractables led to a lower concentration of faradiol esters in the extracts. The HPLC fingerprints of the extracts, the yields of total extract and the concentration of faradiol esters obtained with analytical and pilot scale extractors under the same conditions were comparable.     

Antimicrobial activity of Brazilian propolis extracts against rumen bacteria in vitro

The antimicrobial activity of three Brazilian propolis extracts was evaluated on bacterial strains representing major rumen functional groups. The extracts were prepared using different concentrations of propolis and alcohol, resulting in different phenolic compositions. The propolis extracts inhibited the growth of Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, Prevotella albensis M384, Peptostreptococcus sp. D1, Clostridium aminophilum F and Streptococcus bovis Pearl11, while R. albus 20, Prevotella bryantii B₁4 and Ruminobacter amylophilus H18 were resistant to all the extracts. The inhibited strains showed also different sensitivity to propolis; the hyper-ammonia-producing bacteria (C. aminophilum F and Peptostreptococcus sp. D1) being the most sensitive. Inhibition of hyper-ammonia-producing bacteria by propolis would be beneficial to the animal. The extract containing the lowest amount of phenolic compounds (LLOS C3) showed the lowest antimicrobial activity against all the bacteria. The major phenolic compounds identified in the propolis extracts (naringenin, chrysin, caffeic acid, p-coumaric acid and Artepillin C) were also evaluated on four sensitive strains. Only naringenin showed inhibitory effect against all strains, suggesting that naringenin is one of the components participating to the antibacterial activity of propolis.     

Inhibition of the Ethanol-induced Potentiation of α1 Glycine Receptor by a Small Peptide That Interferes with Gβγ Binding

Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through Gβγ interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQH_C7), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of Gβγ on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQH_C7 displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with Gβγ or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ∼350Ų involving aspartic acids 186, 228, and 246 in Gβγ where we propose that RQH_C7 binds and exerts its blocking action on the effect of ethanol in GlyR.     

β-Carotene extraction from Dunaliella salina by supercritical CO2

This paper reports the results of supercritical carbon dioxide (scCO₂) extraction of β-carotene from Dunaliella salina as potential alternative to conventional organic solvent extraction. In pilot-scale scCO₂ experiments, the pressure, temperature, and co-solvent concentration were varied. The supercritical extraction at 500 bar, 70 °C, and 10 wt% ethanol as co-solvent yielded in the highly efficient pigment recovery of over 90%. Techno-economic assessment demonstrated higher energy consumption for the scCO₂ extraction that was compensated by lower solvent costs. Thus, comparable pigment production costs to the reference extraction with n-hexane were estimated for the scCO₂ process. Due to the green solvent properties of scCO₂ and ethanol, this approach is highly promising for extraction of algal biomass in industrial scale.

     

Effect of Australian Propolis from Stingless Bees (Tetragonula carbonaria) on Pre-Contracted Human and Porcine Isolated Arteries

Bee propolis is a mixture of plant resins and bee secretions. While bioactivity of honeybee propolis has been reported previously, information is limited on propolis from Australian stingless bees (Tetragonula carbonaria). The aim of this study was to investigate possible vasomodulatory effects of propolis in KCl-precontracted porcine coronary arteries using an ex vivo tissue bath assay. Polar extracts of propolis produced a dose-dependent relaxant response (EC₅₀=44.7±7.0 μg/ml), which was unaffected by endothelial denudation, suggesting a direct effect on smooth muscle. Propolis markedly attenuated a contractile response to Ca²⁺ in vessels that were depolarised with 60 mM KCl, in Ca²⁺-free Krebs solution. Propolis (160 µg/ml) reduced vascular tone in KCl pre-contracted vessels to near-baseline levels over 90 min, and this effect was partially reversible with 6h washout. Some loss in membrane integrity, but no loss in mitochondrial function was detected after 90 min exposure of human cultured umbilical vein endothelial cells to 160 µg/ml propolis. We conclude that Australian stingless bee (T. carbonaria) propolis relaxes porcine coronary artery in an endothelial-independent manner that involves inhibition of voltage-gated Ca²⁺ channels. This effect is partially and slowly reversible upon washout. Further studies are required to determine the therapeutic potential of Australian stingless bee propolis for conditions in which vascular supply is compromised.     

Interaction of Artepillin C with model membranes: Effects of pH and ionic strength

Artepillin C is the major constituent of green propolis, one of the most consumed products in popular medicine owing to its therapeutic effects, including antitumor activity. Artepillin C differs from other cinnamic acid derivatives due to the presence of two prenylated groups in its structure, believed to enhance access to the cell membrane and resulting in pharmacological activity. The membrane outer leaflet of tumor cells is exposed to an acidic extracellular environment, which could modulate the protonation state of antitumor drugs and hence their interaction with the cell membrane. Herein, we investigated the interaction of Artepillin C with Langmuir monolayers and giant unilamellar vesicles (GUVs) of 1,2?dipalmitoyl?sn?glycerol?3?phosphocholine (DPPC) used as model membranes, in physiological and acidic environments. We observed that protonation of the carboxyl group of Artepillin C is essential for the interaction, with larger shifts induced in the surface pressure isotherms of DPPC monolayers in comparison with deprotonated Artepillin C. Also observed was a decrease in lipid packing inferred from the compressibility modulus and Brewster angle microscopy (BAM) images for monolayers on acidic subphases. Results with microscopy techniques on GUVs confirmed that.Artepillin C causes a curvature stress of the lipid bilayer only in its neutral state, causing the GUVs to burst. The stronger effects of neutral Artepillin C on both monolayers and GUVs were maintained when the ionic strength was increased. Taken together, the results indicate that Artepillin C may have preferential attachment to a more acidic environment which might be an important feature for its antitumor activity.     

Optimization of Ultrasonic-Assisted Extraction and Radical-Scavenging Capacity of Phenols and Flavonoids from Clerodendrum cyrtophyllum Turcz Leaves

Ultrasonic-assisted extraction (UAE) was developed to extract phenolic and flavonoid antioxidants from Clerodendrum cyrtophyllum Turcz leaves. The optimal experimental parameters for antioxidant extraction from C. cyrtophyllum leaves were measured using single-factor experimentation combined with response surface methodology (RSM). Total phenolic content (TPC) and total flavonoid content (TFC) assays were used to quantify antioxidant compounds. Next, antioxidant radical scavenging capacity was measured using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) radicals. Optimized extraction conditions for UAE from C. cyrtophyllum leaves were as follows: 60.9% ethanol, 85.4 min, and 63.3°C for maximal TPC extraction (16.8±0.2 mg GAE/g DW); 67.7% ethanol, 82.9 min, and 63.0°C for maximal TFC extraction (49.3±0.4 mg RT/g DW); 48.8% ethanol, 85.1 min, and 63.9°C for maximal DPPH radical-scavenging capacity (86.8±0.2%); and 50.6% ethanol, 81.3 min, and 63.4°C for maximal ABTS radical-scavenging capacity (92.9±0.5%). Ethanol concentration was the most important factor in the extraction process. Our work offers optimal extraction conditions for C. cyrtophyllum as a potential source of natural antioxidants.     

Antioxidative bioavailability of artepillin C in Brazilian propolis

Propolis has strong antioxidative activity. We investigated here whether this activity was available in intestinal Caco-2 and hepatic HepG2 cells. Phenolics in Brazilian propolis, extracted with ethyl acetate after the removal of resin and wax with 90% methanol, included artepillin C at 21 mmol/100 g, p-coumaric acid and cinnamic acid relatives 24mmol, kaempferol and its derivatives 9.4 mmol, naringenin 2.8 mmol, isosakuranetin 0.9 mmol, chrysin at 0.8 mmol/100 g, and several minor components. When the extract was added to the apical side of Caco-2 monolayers, artepillin C was specifically incorporated into the cells and released to the basolateral side mostly without conjugation. Then, artepillin C was added to HepG2 cells and exposed to reactive oxygens. Artepillin C prevented oxidative damage dose-dependently, and suppressed lipid peroxidation evaluated with thiobarbituric acid reactive substances by 16% and the formation of 8-hydroxy-2'-deoxyguanosine in DNA by 36% at a concentration of 20microM. Artepillin C is a bioavailable antioxidant.     

Supercritical Carbon Dioxide Extraction of Allium ursinum: Impact of Temperature and Pressure on the Extracts Chemical Profile

The aim of this study was to extract Allium ursinum L. for the first time by supercritical carbon dioxide (SC−CO₂) as green sustainable method. The impact of temperature in the range from 40 to 60 °C and pressure between 150 and 400 bar on the quality of the obtained extracts and efficiency of the extraction was investigated. The highest extraction yield (3.43 %) was achieved by applying the extraction conditions of 400 bar and 60 °C. The analysis of the extracts was performed by gas chromatography and mass spectrometry (GC/MS). The most dominant sulfur-containing constituent of the extracts was allyl methyl trisulfide with the highest abundance at 350 bar and 50 °C. In addition, the presence of other pharmacologically potent sulfur compounds was recorded including S-methyl methanethiosulfinate, diallyl trisulfide, S-methyl methylthiosulfonate, and dimethyl trisulfide. Multivariate data analysis tool was utilized to investigate distributions of the identified compounds among the extracts obtained under various extraction conditions and yields. It was determined that the SC−CO₂ extraction can by efficiently used for A. ursinum.     

Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature

Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37°C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33°C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to ~1.5-fold higher expression of green fluorescent protein (GFP) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By following the time course of the GFP-expressing cells growing at 37°C and 33°C from 24 hours after transfection (including 19 hours recovery at 37°C in the normal growth medium), we found that a mild hypothermia (i.e., 33°C) reduces the growth rate of HEK-293S cells, while increasing cellular productivity of recombinant proteins. As a result, green cells remain undivided in a longer period of time. Not surprisingly, the property of a recombinant protein expressed in the cells grown at 33°C is unaffected, as shown by the use of AMPA receptors. We further demonstrate with the use of PC12 cells that this method may be especially useful when a recombinant protein is difficult to express using a chemical-based, transient transfection method.     

蜂胶活性提取物抑菌效果的研究

采用超声波乙醇浸提法和超临界CO_2萃取法制备蜂胶活性提取物,分别以70%乙醇、丙三醇、聚乙二醇400作为溶剂将蜂胶活性提取物制成溶液.并通过超声波和加热方式对蜂胶活性提取物溶液进行预处理,观察其对常见食品致病菌的抑菌和延长鲜牛肉的保鲜期效果.结果表明:两种蜂胶活性提取物溶液对大肠杆菌、金黄色葡萄球菌、枯草杆菌、蜡样芽孢杆菌、黑曲霉、产黄青霉均有不同程度的抑菌效果,其中对金黄色葡萄球菌、蜡样芽孢杆菌抑菌效果较好.同时发现两者都能较好地延长鲜牛肉的保鲜期.     

Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

How Does Brazilian Green Propolis Act in Normal and Tumor Cells? Identification of Compounds in or out Monocytes or HEp-2 Cells

The aim of this study was to identify propolis compounds after incubation of normal and tumor cells (monocytes and HEp-2 cells, respectively) with Brazilian green propolis, in the lysate and supernatant of cell cultures and within these cells by gas chromatography-mass spectrometry (GC/MS). Cinnamic acid derivatives were generally localized in the lysate of both cell lines after incubation, suggesting these compounds are actively transported across the membrane into the cytoplasm. Terpenes were also found in the lysate. Artepillin C, in contrast, was localised only in the supernatant. Some constituents were unobservable after incubation, especially in monocytes, suggesting the compounds had been degraded. Our findings shed light on the possible sites of action (intracellular or via a cell membrane protein) and the bioavailability of various constituents of propolis, as well as possible modes of delivery of bioactive constituents.     

Inactivation of a Norovirus by High-Pressure Processing

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20°C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log₁₀ PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5°C; a 5-min pressure treatment of 350 MPa at 30°C inactivated 1.15 log₁₀ PFU of virus, while the same treatment at 5°C resulted in a reduction of 5.56 log₁₀ PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5°C and 20°C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log₁₀ PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.     

Free radical scavenging of grape pomace extracts from Cabernet sauvingnon (Vitis vinifera)

Pressed grape pomace obtained from the wine production of Cabernet sauvignon (Vitis vinifera) vintage was dried until 9.8% moisture content, ground and submitted to extraction of soluble components from different extraction techniques. Low pressure extractions were performed with ethanol maceration followed by fractionation with n-hexane, dichloromethane, butanol and ethyl acetate. These solvents were furthermore applied for soxhlet extraction. Supercritical fluid extraction (SFE) was also performed to obtain grape pomace extracts by using pure CO(2) and CO(2) with ethanol as co-solvent in concentrations of 10, 15 and 20%w/w. The operating condition used in high pressure extractions was 150bar and 40 degrees C. The antioxidant activity of the grape pomace extracts was determined considering the free radical scavenging assay using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and was correlated with the total phenol content determined according to the Folin-Ciocalteu method. The results obtained in DPPH tests indicate the highest antioxidant activity of 96.6+/-0.3%AA, with an IC(50) value of 13+/-1, for the extracts obtained with ethyl acetate in solid-liquid extraction. The highest yield values were achieved in soxhlet extraction with ethanol (13.2%w/w) and with butanol (12.2%w/w), and also by SFE with 15% ethanol (9.2%w/w). The lipophilic composition of grape pomace extracts was evaluated by gas chromatography-mass spectrometry with the identification of components like linoleic acid and ethyl linoleate, with important therapeutic activities.