Potent inhibitory effects of N-aryl S-alkylthiocarbamate derivatives on the dopa oxidase activity of mushroom tyrosinase

This study reports the potent inhibitory effect of N-aryl S-alkylthiocarbamate derivatives on mushroom tyrosinase (MT) activity. N-Aryl S-alkylthiocarbamate derivatives were found to exhibit a potent inhibitory effect on the dopa (3,4-dihydroxyphenylalanine) oxidase activity of mushroom tyrosinase. Most of the N-aryl S-alkylthiocarbamate derivatives (compounds from A to J) exhibited higher inhibitory effects than kojic acid (IC50=318 microM), a well known tyrosinase inhibitor. Tyrosinase was the most inhibited by S-phenetyl N-phenylthiocarbamate (compound E, IC50=7.25 microM), and this inhibition was 44 times stronger than that of kojic acid. Compound E exhibited 95.0% of inhibition at 100 microM. A kinetic study of MT inhibition by compound E using the Lineweaver-Burk plots analysis was performed. And the kinetics profiles observed suggest that compound E competitively inhibits MT.     

Novel Anti-Melanogenic Compounds, (Z)-5-(Substituted Benzylidene)-4-thioxothiazolidin-2-one Derivatives: In Vitro and In Silico Insights

To confirm that the β-phenyl-α,β-unsaturated thiocarbonyl (PUSTC) scaffold, similar to the β-phenyl-α,β-unsaturated carbonyl (PUSC) scaffold, acts as a core inhibitory structure for tyrosinase, twelve (Z)-5-(substituted benzylidene)-4-thioxothiazolidin-2-one ((Z)-BTTZ) derivatives were designed and synthesized. Seven of the twelve derivatives showed stronger inhibitory activity than kojic acid against mushroom tyrosinase. Compound 2b (IC₅₀ = 0.47 ± 0.97 µM) exerted a 141-fold higher inhibitory potency than kojic acid. Kinetic studies’ results confirmed that compounds 2b and 2f are competitive tyrosinase inhibitors, which was supported by high binding affinities with the active site of tyrosinase by docking simulation. Docking results using a human tyrosinase homology model indicated that 2b and 2f might potently inhibit human tyrosinase. In vitro assays of 2b and 2f were conducted using B16F10 melanoma cells. Compounds 2b and 2f significantly and concentration-dependently inhibited intracellular melanin contents, and the anti-melanogenic effects of 2b at 10 µM and 2f at 25 µM were considerably greater than the inhibitory effect of kojic acid at 25 µM. Compounds 2b and 2f similarly inhibited cellular tyrosinase activity and melanin contents, indicating that the anti-melanogenic effects of both were due to tyrosinase inhibition. A strong binding affinity with the active site of tyrosinase and potent inhibitions of mushroom tyrosinase, cellular tyrosinase activity, and melanin generation in B16F10 cells indicates the PUSTC scaffold offers an attractive platform for the development of novel tyrosinase inhibitors.     

Microwave-assisted synthesis and biological evaluation of new thiazolylhydrazone derivatives as tyrosinase inhibitors and antioxidants

In this work, we have synthesized a series of 2-thiazolylhydrazone derivatives ( 1–27 ) and investigated their biological activities as tyrosinase inhibitors and antioxidants. Some compounds showed potent tyrosinase inhibitory activities and 4-(2-(2-(1-(4-Aminophenyl)ethylidene)-hydrazinyl)thiazol-4-yl) phenol ( 26 ) showed more potent inhibitory effect than the standard tyrosinase inhibitor kojic acid (IC₅₀: 9.8 μM vs. 23.6 μM). Compounds 2 , 14 , and 26 exhibited high antioxidant activities in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The structure–activity relationship (SAR) indicated that the substitutions of bromine, hydroxyl group, and amino groups cause great effect to the inhibition effect against tyrosinase. The mechanism and kinetic studies demonstrated that the inhibitory effect of compound 26 on the tyrosinase by acting as the reversible and uncompetitive inhibitor. Docking studies suggests that compound 26 interacts strongly with mushroom tyrosinase via hydrogen bonding.     

Regulation of melanin synthesis by selenium-containing carbohydrates

This study reports depigmenting potency of selenium-containing carbohydrates, which would be based upon the finding of direct inhibition to mushroom tyrosinase. Two selenoglycosiede, SG-3 (bis(2,3,4-tri-O-acetyl-beta-D-arabinopyranosyl) selenide) and SG-8 (4'-methylbenzoyl 2,3,4,6-tetra-O-acetyl-D-selenomanopyranoside) among eleven selenium-containing compounds examined, were discovered to be effective depigmenting compounds on a mushroom tyrosinase inhibitory assay. SG-3 exhibited a competitive inhibition effect that was similar to kojic acid, well-known tyrosinase inhibitor. At 100 microM and 150 microM, SG-8 had an uncompetitive inhibitory effect that was higher than kojic acid. A study of a melan-a cell originated-tyrosinase inhibition assay showed that SG-8 had a lower inhibitory effect than kojic acid. SG-3 showed a similar inhibition effect to kojic acid on the melan-a cell-originated tyrosinase inhibitory assay. SG-8 showed dose-dependently cytotoxicity in a study of inhibition melanin synthesis by melan-a cells. Most melan-a cells did not survive after being treated with 20 microM of SG-8. At 10 microM, SG-3 inhibited melanin synthesis in the melan-a cells, and the effect was similar to phenylthiourea, which is a well-known inhibitor of melanin synthesis. Therefore, SG-3 is a new candidate for depigmenting reagents.     

Methoxy-Substituted Tyramine Derivatives Synthesis,Computational Studies and Tyrosinase Inhibitory Kinetics

Targeting tyrosinase for melanogenesis disorders is an established strategy. Hydroxyl-substituted benzoic and cinnamic acid scaffolds were incorporated into new chemotypes that displayed in vitro inhibitory effects against mushroom and human tyrosinase for the purpose of identifying anti-melanogenic ingredients. The most active compound 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2,4-dihydroxyphenyl) acrylate (Ph9), inhibited mushroom tyrosinase with an IC₅₀ of 0.059 nM, while 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) had an IC₅₀ of 2.1 nM compared to the positive control, kojic acid IC₅₀ 16700 nM. Results of human tyrosinase inhibitory activity in A375 human melanoma cells showed that compound (Ph9) and Ph6 exhibited 94.6% and 92.2% inhibitory activity respectively while the positive control kojic acid showed 72.9% inhibition. Enzyme kinetics reflected a mixed type of inhibition for inhibitor Ph9 (K_i 0.093 nM) and non-competitive inhibition for Ph6 (K_i 2.3 nM) revealed from Lineweaver–Burk plots. In silico docking studies with mushroom tyrosinase (PDB ID:2Y9X) predicted possible binding modes in the catalytic site for these active compounds. Ph9 displayed no PAINS (pan-assay interference compounds) alerts. Our results showed that compound Ph9 is a potential candidate for further development of tyrosinase inhibitors.     

Inhibitory Effects of Coumarin Derivatives on Tyrosinase

In this study, a series of coumarin derivatives were synthesized and their inhibitory effects on the activity of mushroom tyrosinase were evaluated. As a result of measuring the inhibition of tyrosinase activity of these derivatives, the compounds 3e (1.05 μM), 3f (0.83 μM), 3h (0.85 μM), 3i (1.05 μM), and 3k (0.67 μM) of the geranyloxycoumarin derivatives were highly active at a concentration of 0.8%. The geranyloxycoumarin derivatives exhibited better activity than the hydroxycoumarin derivatives. Among the geranyloxycoumarin derivatives, compound 3k was two times more active than arbutin, a positive control, at a concentration of 0.4%. The above results suggest that geranyloxycoumarin derivatives have great potential for application as functional cosmetic ingredients with tyrosinase-inhibiting activity.     

Biological and Cheminformatics Studies of Newly Designed Triazole Based Derivatives as Potent Inhibitors against Mushroom Tyrosinase

A series of nine novel 1,2,4-triazole based compounds were synthesized through a multistep reaction pathway and their structures were scrutinized by using spectral methods such as FTIR, LC-MS, 1H NMR, and 13C NMR. The synthesized derivatives were screened for inhibitory activity against the mushroom tyrosinase and we found that all the synthesized compounds demonstrated decent inhibitory activity against tyrosinase. However, among the series of compounds, N-(4-fluorophenyl)-2-(5-(2-fluorophenyl)-4-(4-fluorophenyl)-4H-1,2,4-triazol-3-ylthio) acetamide exhibited more prominent activity when accompanied with the standard drug kojic acid. Furthermore, the molecular docking studies identified the interaction profile of all synthesized derivatives at the active site of tyrosinase. Based on these results, N-(4-fluorophenyl)-2-(5-(2-fluorophenyl)-4-(4-fluorophenyl)-4H-1,2,4-triazol-3-ylthio) acetamide could be used as a novel scaffold to design some new drugs against melanogenesis.     

胀果甘草提取物对蘑菇酪氨酸酶的抑制作用

筛选胀果甘草是对蘑菇酪氨酸酶抑制活性最强的提取物,并研究其对蘑菇酪氨酸酶的抑制类型,探究其抑制作用机理。 考察了胀果甘草7种不同溶剂包括甘草酸、提酸废液、石油醚、氯仿、乙酸乙酯、正丁醇及水提取物对蘑菇酪氨酸酶的抑制作用和对2,2-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐(ABTS)自由基阳离子(ABTS·﹢)、羟基自由基(HO·)的清除作用,根据双倒数曲线图形判断对蘑菇酪氨酸酶的抑制作用类型,结合抗氧化能力探究对蘑菇酪氨酸酶的抑制作用机理。 在胀果甘草7种溶剂提取物中,以乙酸乙酯提取物对蘑菇酪氨酸酶具有最强的抑制作用,IC50为3.4775 g/L,双倒数曲线做图得到了一组纵轴截距不变的曲线,抑制常数K1为0.6667 g/L,胀果甘草乙酸乙酯提取物也具有最强的清除ABTS·﹢、HO·的能力,半清除浓度和速率常数分别为0.0442 g/L和4.634×108 L/(g·s)。 胀果甘草乙酸乙酯提取物对蘑菇酪氨酸酶的抑制作用是可逆竞争性抑制,推测其对酪氨酸酶的抑制是通过清除了氧自由基和作为竞争性底物而实现的。     

Identification of Mushroom and Murine Tyrosinase Inhibitors from Achillea biebersteinii Afan. Extract

Growing scientific evidence indicates that Achillea biebersteinii is a valuable source of active ingredients with potential cosmetic applications. However, the data on its composition and pharmacological properties are still insufficient. This study aims to optimize the extraction procedure of the plant material, evaluate its phytochemical composition, and compare anti-tyrosinase potential of A. biebersteinii extracts obtained by various methods. In order to identify compounds responsible for the tyrosinase inhibitory activity of A. biebersteinii, the most active anti-tyrosinase extract was fractionated by column chromatography. The fractions were examined for their skin lightening potential by mushroom and murine tyrosinase inhibitory assays and melanin release assay. HPLC-ESI-Q-TOF-MS/MS analysis of the total extract revealed the presence of several phenolic acids, flavonoids, flavonoid glucosides, and carboxylic acid. Among them, fraxetin-8-O-glucoside, quercetin-O-glucopyranose, schaftoside/isoschaftoside, gmelinin B, 1,3-dicaffeoylquinic acid (1,3-DCQA), and ferulic acid were found in the fractions with the highest skin lightening potential. Based on obtained qualitative and quantitative analysis of the fractions, it was assumed that the caffeoylquinic acid derivatives and dicaffeoylquinic acid derivatives are more likely responsible for mushroom tyrosinase inhibitory activity of A. biebersteinii extracts and fractions. Ferulic acid was proposed as the most active murine tyrosinase inhibitor, responsible also for the reduced melanin release from B16F10 murine melanoma cells.     

Selenium-sulfur analogs. 4. Synthesis and characterization of (±)-β-(2-amino-1,3-selenazol-4-yl)alanine

The novel amino acid (±)-β-(2-amino-1,3-selenazol-4-yl)alanine 4a was prepared from selenourea and 1,3-dichloropropan-2-one via a four-step synthetic sequence. Nuclear magnetic resonance analysis indicated a downfield chemical shift of δ 0.29 to 0.30 and δ 0.57 for the C₅-proton of the protonated 2-amino-and 2-acetamido-1,3-selenazoles respectively compared with the analogous thiazoles. The infrared spectra of the selenazole and corresponding thiazole compounds were virtually identical.     

Inhibitory Effects of Fatty Acids on the Activity of Mushroom Tyrosinase

The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.     

Synthesis and Preliminary In Vitro Biological Evaluation of 5-Chloro-2-(Substituted Phenyl)Benzo[d]Thiazole Derivatives Designed As Novel Antimelanogenesis Agents

We describe the design, synthesis, and biological activities of 5-chloro-2-(substituted phenyl)benzo[d]thiazole derivatives as novel tyrosinase inhibitors. Among them, 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) and 2-bromo-4-(5-chloro-benzo[d]thiazol-2-yl)phenol (MHY966) showed inhibitory activity higher than or similar to kojic acid, against mushroom tyrosinase. Therefore, we carried out kinetic studies on the two compounds with potent tyrosinase inhibitory effects. Kinetic analysis of tyrosinase inhibition revealed that all of these compounds are competitive inhibitors. MHY884 and MHY966 effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with ??-melanocyte stimulating hormone (??-MSH). These data strongly suggest that the newly synthesized compounds MHY884 and MHY966 could suppress production of melanin via inhibition of tyrosinase activity.     

Development of 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one as a novel whitening agent

A stable derivative of kojic acid, 5-[(3-aminopropyl)phosphinooxy]-2-(hydroxymethyl)-4H-pyran-4-one (Kojyl-APPA), was synthesized in good yield. The effects of Kojyl-APPA on tyrosinase activity and melanin synthesis were investigated. Kojyl-APPA showed tyrosinase inhibition effect (30%) in situ, but not in vitro. Kojyl-APPA inhibited tyrosinase activity significantly at 24 h after treatment in normal human melanocytes. It means that Kojyl-APPA is not a direct inhibitor of tyrosinase itself, but it is converted to a potential inhibitor kojic acid enzymatically in cells. In addition, Kojyl-APPA decreased melanin content to 75% of control in melanoma cells and decreased neomelanin synthesis to 43% of control in normal human melanocytes. Its permeation through skin increased by about 8 times as compared with kojic acid.     

Kinetics of Ergothioneine Inhibition of Mushroom Tyrosinase

The native amino acid ergothioneine, a thiourea derivative of histidine, inhibits mushroom tyrosinase activity in a dose-dependent manner, with an IC₅₀ value of 1.025 mg/ml (4.47 mM). By contrast, histidine exhibited no inhibitory effect on mushroom tyrosinase activity. We characterized ergothioneine as a noncompetitive tyrosinase inhibitor using a Lineweaver–Burk plot of experimental kinetic data. The IC₅₀ value for ergothioneine scavenging of 2,2-diphenyl-1-picrylhydrazyl was 6.110 ± 0.305 mg/ml, much higher than the IC₅₀ for inhibition of tyrosinase activity which indicating ergothioneine on tyrosinase shows a weak correlation to its antioxidative activity. The results demonstrated that ergothioneine has a potent inhibition effect on tyrosinase enzyme activity, resulting from the presence of the sulfur substituted imidazole ring in ergothioneine.     

A Novel Heptapeptide with Tyrosinase Inhibitory Activity Identified from a Phage Display Library

Peptidic inhibition of the enzyme tyrosinase, responsible for skin pigmentation and food browning, would be extremely useful for the food, cosmetics, and pharmaceutical industries. In order to identify novel inhibitory peptides, a library of short sequence oligopeptides was screened to reveal direct interaction with the tyrosinase. A phage displaying heptapeptide (IQSPHFF) was found to bind most strongly to tyrosinase. The inhibitory activity of the heptapeptide was evaluated using mushroom tyrosinase. The results showed that the peptide inhibited both the monophenolase and diphenolase activities of mushroom tyrosinase with IC₅₀ values of 1.7 and 4.0 mM, respectively. The heptapeptide is thought to be a reversible competitive inhibitor of diphenolase with the inhibition constants (Ki) of 0.765 mM. To further investigate how the heptapeptide exerts its inhibitory effect, a docking study between tyrosinase and heptapeptide was performed. The simulation showed that the heptapeptide binds in the active site of the enzyme near the catalytically active Cu ions and forms hydrogen bonds with five histidine residues on the active site. Phage display technology is thus a useful approach for the screening of potential tyrosinase inhibitors and could be widely applicable to a much wider range of enzymes.     

Kinetic Evaluation of Aminoethylisothiourea on Mushroom Tyrosinase Activity

This study demonstrates that aminoethylisothiourea (AET), a potent inhibitor of inducible nitric oxide synthase, is an irreversible competitive inhibitor of mushroom tyrosinase by chelation to the active site of tyrosinase when l-3,4-dihydroxyphenylalanine was assayed spectrophotometrically. The spectrophotometric recordings of the inhibition of tyrosinase by AET were characterized by the presence of a lag period prior to the attainment of an inhibited steady-state rate. The lag period corresponded to the time in which AET was reacting with the enzymatically generated o-quinone. Increasing AET concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady-state rate were dependent on AET, substrate, and tyrosinase concentrations. The inhibition of diphenolase activity of tyrosinase by AET showed positive kinetic cooperativity which arose from the protection of both substrate and o-quinone against inhibition by AET. The UV-visible spectrum of a mixture of tyrosinase and AET exhibited a characteristic shoulder peak ascribed to the chelation of AET to the active site of tyrosinase. Moreover, the presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition when CuSO₄ was added to the assay medium on tyrosinase activity.     

Syntheses of arbutin-alpha-glycosides and a comparison of their inhibitory effects with those of alpha-arbutin and arbutin on human tyrosinase

The effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) and 4-hydroxyphenyl beta-glucopyranoside (arbutin) on the activity of tyrosinase from human malignant melanoma cells were examined. The inhibitory effect of alpha-arbutin on human tyrosinase was stronger than that of arbutin. The K(i) value for alpha-arbutin was calculated to be 1/20 that for arbutin. We then synthesized arbutin-alpha-glycosides by the transglycosylation reaction of cyclomaltodextrin glucanotransferase using arbutin and starch, respectively, as acceptor and donor molecules. The structural analyses using 13C- and 1H-NMR proved that the transglycosylated products were 4-hydroxyphenyl beta-maltoside (beta-Ab-alpha-G1) and 4-hydroxyphenyl beta-maltotrioside (beta-Ab-alpha-G2). These arbutin-alpha-glycosides exhibited competitive type inhibition on human tyrosinase, and their K(i) values were calculated to be 0.7 mM and 0.9 mM, respectively. These arbutin-alpha-glycosides possessed stronger inhibitory activity than arbutin, but less activity than alpha-arbutin. These results suggested that the alpha-glucosidic linkage of hydroquinone-glycosides plays an important role in the inhibitory effect on human tyrosinase.     

两种多酸型酪氨酸酶抑制剂的性能研究

以H₃PW₁₂O₄₀和H₄SiW₁₂O₄₀(简写为PW₁₂和SiW₁₂)为效应物, 测定其对酪氨酸酶活力的抑制作用. 通过非变性聚丙烯凝胶(Native-PAGE)电泳确定酪氨酸酶是多家族基因编码, 其分子量为3×10⁴~3.4×10⁴, 4.2×10⁴~4.6×10⁴, 6.4×10⁴~6.8×10⁴, 且均具有活性, 测定PW₁₂和SiW₁₂对酪氨酸酶的抑制效果, 并结合酶动力学法研究其抑制机理. 结果表明, 当PW₁₂和SiW₁₂浓度分别达到13和25 mmol/L时, 酪氨酸酶的活力完全被抑制, 即PW₁₂和SiW₁₂对酪氨酸酶二酚酶具有不同程度的抑制效果. 当所加酶量为0.0173 mg/mL时, PW₁₂和SiW₁₂对酪氨酸酶二酚酶活力的半抑制率 (IC₅₀)分别为1.57和2.31 mmol/L, 它们对酪氨酸酶二酚酶的抑制均为可逆过程. 其中, PW₁₂对二酚酶的抑制类型为混合型, 其K_I及K_IS分别为0.34和0.43 mmol/L, SiW₁₂对二酚酶的抑制类型表现为竞争型, 其K_I为0.59 mmol/L. 综合考虑IC₅₀值和抑制常数等参数, PW₁₂对酪氨酸酶二酚酶的抑制能力优于SiW₁₂.     

Enzyme Modification and Oxidative Cross‐linking Using Carboxylate‐, Phenol‐ and Catechol‐Conjugated 1,8‐Naphthalimides

The ground‐ and excited‐state interactions of β‐alanine, tyrosine and l ‐dopa substituted 1,8 naphthalimides (NI‐Ala, NI‐Tyr and NI‐Dopa) with lysozyme and mushroom tyrosinase were evaluated to understand the mechanism of oxidative modification. Photooxidative cross‐linking of lysozyme was observed for all three conjugates. The yield was significantly reduced for NI‐Tyr and NI‐Dopa due to intramolecular electron transfer to the excited singlet state of the 1,8‐naphthalimide. Incubation of NI‐Tyr and NI‐Dopa with mushroom tyrosinase resulted in an increased fluorescence from the naphthalimide, suggesting that the phenol and catechol portion of the conjugates are oxidized by the enzyme. This result demonstrates that the compounds bind in the active site of mushroom tyrosinase. The catalytic activity of mushroom tyrosinase to oxidize both tyrosine (monophenolase) and l ‐dopa (diphenolase) was modified by NI‐Tyr and NI‐Dopa. Monophenolase activity was inhibited, and the diphenolase activity was enhanced in the presence of these conjugates. Detailed Michaelis–Menten studies show that both V_max and K_m are modified, consistent with a mixed inhibition mechanism. Collectively, the results show that the compounds interact in the enzyme's active site, but also modify the distribution of the enzyme's oxidation states that are responsible for catalysis.     

Biological activities of phenolic compounds isolated from galls of Terminalia chebula Retz. (Combretaceae)

The aqueous extract of galls from Terminalia chebula Retz. (Combretaceae) was fractionated on Diaion and refractionated on octadecyl silica column. Six phenolic compounds were isolated and identified as gallic acid (1), punicalagin (2), isoterchebulin (3), 1,3,6-tri-O-galloyl-β-D-glucopyranose (4), chebulagic acid (5) and chebulinic acid (6). All of the compounds showed stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and melanin inhibitory activities than ascorbic acid, butylated hydroxytoluene, α-tocopherol, arbutin and kojic acid, the reference compounds. Gallic acid (1) exhibited inhibitory activity against nitric oxide production in lipopolysaccharide-activated macrophages. However, all isolated compounds exhibited less activity than the reference compounds in mushroom tyrosinase inhibition and human tumour cytotoxicity assays. This study has demonstrated that the phenolic compounds isolated from galls of T. chebula might contribute significantly due to their antioxidant and whitening activities.