Computational prediction of protein ubiquitination sites mapping on Arabidopsis thaliana

Among the protein post-translational modifications (PTMs), ubiquitination is considered as one of the most significant processes which can regulate the cellular functions and various diseases. Identification of ubiquitination sites becomes important for understanding the mechanisms of ubiquitination-related biological processes. Both experimental and computational approaches are available for identifying ubiquitination sites based on protein sequences of different species. The experimental approaches are time-consuming, laborious and costly. In silico prediction is an alternative time saving, easier and cost-effective approach for identifying ubiquitination sites. Moreover, the sequence patterns in the different species around the ubiquitination sites are not similar which demands species-specific predictors. Therefore, in this study, we have proposed a novel computational method for identifying ubiquitination sites based on protein sequences of A. thaliana species which will be robust against outlying observations also. Through the comparative study of two encoding schemes and three classifiers, the random forest (RF) based predictor was selected as the best predictor under the CKSAAP encoding scheme with 1:1 ratio of positive and negative samples (i.e. ubiquitinated and non-ubiquitinated) in training dataset. The proposed predictor produced the area under the ROC curve (AUC score) as 0.91 and 0.86 for 5-fold cross-validation test with the training dataset and the independent test dataset of A. thaliana respectively. The proposed RF based predictor also performed much better than the other existing ubiquitination sites predictors for A. thaliana.     

Evaluation of tyrosinase inhibitory activity in Salak (<Emphasis Type="Italic">Salacca zalacca</Emphasis>) extracts using the digital image-based colorimetric method

A simple toolbox was developed for the evaluation of tyrosinase inhibitory activity in Salak (Salacca zalacca) extracts by the colorimetric measurement based on a photograph taken by a digital camera or a smartphone camera. The reaction of 3,4-dihydroxyphenylalanine (l-DOPA) and tyrosinase was employed to form the dopaquinone dye, which decreases with the increase of the tyrosinase inhibitor. Under the optimum conditions, the Salak extracts were examined for the tyrosinase inhibitory activity. The captured picture of dopaquinone dye product was analyzed by reading blue color intensity using an Adobe Photoshop CS6 program. The tyrosinase inhibition of the extracts was calculated from the blue color intensity, and expressed as %inhibition and IC₅₀ values. The obtained results from the developed method correlated well with those obtained from the microplate reader instrument. The achievement of this research will be a guideline for creating any simple analytical instrument based on colorimetry. In addition, the information on tyrosinase inhibitory activity of the Salak extracts is useful for the application of this fruit to produce the supplement food and the cosmetic in the future.     

Rosa platyacantha Schrenk from Kazakhstan—Natural Source of Bioactive Compounds with Cosmetic Significance

Plants belonging to the Rosa genus are known for their high content of bioactive molecules and broad spectrum of healing and cosmetic activities. Rosa platyacantha Schrenk is a wild-type species abundant in the mountainous regions of Kazakhstan. The phytochemical composition as well as the bioactivity of R. platyacantha extracts have not been fully investigated to date. In this study, various parts of R. platyacantha plant, collected in Almaty region, Kazakhstan, were used to prepare five hydroalcoholic extracts (R1–R5). The extracts were compared for the content of phytochemicals and selected biological activities, which are important for the potential cosmetic application of R. platyacantha. Extract R3, prepared from flower buds, showed the most significant antioxidant and tyrosinase inhibitory potential, decreasing the monophenolase and diphenolase activities of tyrosinase. Extract R3 showed also collagenase inhibitory activity and cytotoxicity against human melanoma cells A375, being less cytotoxic for noncancerous skin keratinocytes HaCaT. Analysis of fractions E and F, obtained from R3 extracts, revealed that quercetin, kaempferol, rutin, and their derivatives are more likely responsible for the tyrosinase inhibitory properties of R. platyacantha extracts.     

An ultrafiltration high-performance liquid chromatography coupled with diode array detector and mass spectrometry approach for screening and characterising tyrosinase inhibitors from mulberry leaves

Tyrosinase is a key enzyme in melanin synthesis. Its inhibitor may be used to efficiently treat hyperpigmentation and widely applied in cosmetic products and food supplements. In the present study, a new assay based on ultrafiltration high-performance liquid chromatography coupled with diode array detector and mass spectrometry (HPLC–DAD–MS) was developed for the rapid screening and identification of ligands for tyrosinase. Experiments were carried out to select the optimal binding conditions, tyrosinase concentration, and incubation time. Non-specific binding to the denatured tyrosinase was also investigated. Twelve compounds with tyrosinase binding activity were found in mulberry leaf extracts. The identities of these compounds were characterised by HPLC–DAD–MSⁿ. Particularly, two compounds, namely, quercetin-3-O-(6-O-malonyl)-β-d-glucopyranoside and kaempferol-3-O-(6-O-malonyl)-β-d-glucopyranoside, were identified as new tyrosinase inhibitors. The screening results were verified by tyrosinase inhibition assays. Experimental results proved that the proposed method could rapidly screen tyrosinase inhibitors in complex mixtures.     

Kinetics and Computational Docking Studies on the Inhibition of Tyrosinase Induced by Oxymatrine

A combination of enzymatic inhibition kinetics and computational prediction was employed to search for an effective inhibitor of tyrosinase. We found that oxymatrine significantly inhibited tyrosinase, and that this reaction was not accompanied by detectable conformational changes. Kinetic analysis showed that oxymatrine reversibly inhibited tyrosinase in a mixed-type manner. Measurements of intrinsic and ANS-binding fluorescences showed that oxymatrine did not induce any conspicuous changes in the tertiary structure. We also conducted a docking simulation between tyrosinase and oxymatrine using two docking programs, Dock6.3 and AutoDock4.2 (binding energy was ?118.81 kcal/mol for Dock6 and ?8.04 kcal/mol for AutoDock4). The results also suggested that oxymatrine interacts mostly with the residues of CYS83 and HIS263 in the active site of tyrosinase. This strategy of predicting tyrosinase inhibition by simulation of docking coupling with kinetics may prove useful in screening for potential tyrosinase inhibitors. Knowledge of tyrosinase inhibition can provide medical, cosmetic, and agricultural applications. Our study suggests that oxymatrine is an important agent for various applications related to pigment formation.     

The Hypopigmentation Mechanism of Tyrosinase Inhibitory Peptides Derived from Food Proteins: An Overview

Skin hyperpigmentation resulting from excessive tyrosinase expression has long been a problem for beauty lovers, which has not yet been completely solved. Although researchers are working on finding effective tyrosinase inhibitors, most of them are restricted, due to cell mutation and cytotoxicity. Therefore, functional foods are developing rapidly for their good biocompatibility. Food-derived peptides have been proven to display excellent anti-tyrosinase activity, and the mechanisms involved mainly include inhibition of oxidation, occupation of tyrosinase’s bioactive site and regulation of related gene expression. For anti-oxidation, peptides can interrupt the oxidative reactions catalyzed by tyrosinase or activate an enzyme system, including SOD, CAT, and GSH-Px to scavenge free radicals that stimulate tyrosinase. In addition, researchers predict that peptides probably occupy the site of the substrate by chelating with copper ions or combining with surrounding amino acid residues, ultimately inhibiting the catalytic activity of tyrosinase. More importantly, peptides reduce the tyrosinase expression content, primarily through the cAMP/PKA/CREB pathway, with PI3K/AKT/GSK3β, MEK/ERK/MITF and p38 MAPK/CREB/MITF as side pathways. The objective of this overview is to recap three main mechanisms for peptides to inhibit tyrosinase and the emerging bioinformatic technologies used in developing new inhibitors.     

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.     

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.     

Validation of a High-Performance Liquid Chromatography with Photodiode Array Detection Method for the Separation and Quantification of Antioxidant and Skin Anti-Aging Flavonoids from Nelumbo nucifera Gaertn. Stamen Extract

Nelumbo nucifera Gaertn., or the so-called sacred lotus, is a useful aquatic plant in the Nelumbonaceae family that has long been used to prepare teas, traditional medicines as well as foods. Many studies reported on the phytochemicals and biological activities of its leaves and seeds. However, to date, only few studies were conducted on its stamen, which is the most important ingredient for herbal medicines, teas and other phytopharmaceutical products. Thus, this present study focuses on the following: (1) the application of high-performance liquid chromatography with photodiode array detection for a validated separation and quantification of flavonoids from stamen; (2) the Nelumbo nucifera stamen’s in vitro and in cellulo antioxidant activities; as well as (3) its potential regarding the inhibition of skin aging enzymes for cosmetic applications. The optimal separation of the main flavonoids from the stamen ethanolic extract was effectively achieved using a core-shell column. The results indicated that stamen ethanolic extract has higher concentration of in vitro and in cellulo antioxidant flavonoids than other floral components. Stamen ethanolic extract showed the highest protective effect against reactive oxygen/nitrogen species formation, as confirmed by cellular antioxidant assay using a yeast model. The evaluation of potential skin anti-aging action showed that the stamen extract has higher potential to inhibit tyrosinase and collagenase compared with its whole flower. These current findings are the first report to suggest the possibility to employ N. nucifera stamen ethanolic extract as a tyrosinase and collagenase inhibitor in cosmetic applications, as well as the utility of the current separation method.     

Mushroom tyrosinase inhibition activity of some chromones

Currently, aloesin is used in the cosmetic industry as a whitening agent because it inhibits tyrosinase activity. Aloesin is a C-glycosylated chromone compound isolated from aloe, and it is difficult to synthesize because of C-glycosyl moiety in the molecule. The purpose of this study is to search for a new chromone compound which is easy to synthesize and which posesses stronger tyrosinase inhibitory activity than aloesin. Fourteen chromone derivatives were synthesized and screened for their mushroom-tyrosinase inhibitory activity. 5-Methyl-7-methoxy-2-(2'-benzyl-3'-oxobutyl)chromone (15) showed the strongest activity among tested compounds. Its activity was not only stronger than aloesin, but also stronger than arbutin and kojic acid. The kinetic analysis revealed a competitive inhibition of 15 with tyrosinase for the L-tyrosine binding site.     

Development of nanoformulation for hyperpigmentation disorders: Experimental evaluations,in vitro efficacy and in silico molecular docking studies

Hyperpigmentation is a crucial dermatological disorder. This study aims to formulate a nanoemulsion formulation containing chlorogenic acid (CA) for hyperpigmentation treatment, to carry out characterization studies, and to investigate its efficacy and safety in vitro and in silico analysis.In line with this purpose, CA nanoemulsions (CA-NEs) were developed using the ultrasonic homogenization method. Accelerated stability tests were performed to examine the kinetic and thermodynamic stability of the CA-NEs to ascertain the presence of any stability issues. After the heating–cooling test, appropriate CA-NEs were stored for 60 days in three different stability environments to examine the physicochemical stability and determine the finalized formulation. The toxicity of the finalized CA-NE formulation was evaluated by genotoxicity/mutagenicity and cytotoxicity tests. The tyrosinase and melanogenesis activities of the finalized CA-NE formulation were determined on the Melanoma B16F0 cell line. Finally, the molecular docking method was used to reveal interactions of CA that play an essential role in tyrosinase inhibition. Additionally, the mushroom and human tyrosinase enzymes were used to determine the activity of CA. In addition, the comparison study with the molecular docking method was carried out using kojic acid as a reference molecule.In conclusion, the molecular docking study, pharmacokinetic analyses, and in vitro studies showed that F4P1 coded CA-NE formulation might hold promise as an innovative formulation in cosmetic applications such as skin-lightening effects with its high efficacy and safety profile.     

Biochemical Properties of Tyrosinase from Aspergillus terreus and Penicillium copticola; Undecanoic Acid from Aspergillus flavus,an Endophyte of Moringa oleifera,Is a Novel Potent Tyrosinase Inhibitor

Tyrosinase is a copper-containing monooxygenase catalyzing the O-hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine then to dopaquinone that is profoundly involved in melanin synthesis in eukaryotes. Overactivation of tyrosinase is correlated with hyperpigmentation that is metabolically correlated with severe pathological disorders, so, inhibition of this enzyme is the most effective approach in controlling the overproduction of melanin and its hazardous effects. Thus, searching for a powerful, selective inhibitor of human tyrosinase to limit the hyper-synthesis of melanin is a challenge. Unlike the difficulty of overexpression of human tyrosinase, using fungal tyrosinase as a model enzyme to the human one to evaluate the mechanistics of enzyme inhibition in response to various compounds is the most feasible strategy. Thus, the purification of highly catalytic-efficient fungal tyrosinase, exploring a novel inhibitor, and evaluating the mechanistics of enzyme inhibition are the main objectives of this work. Aspergillus terreus and Penicillium copticola were reported as the most potential tyrosinase producers. The biochemical properties suggest that this enzyme displays a higher structural and catalytic proximity to human tyrosinase. Upon nutritional bioprocessing by Plackett–Burman design, the yield of tyrosinase was increased by about 7.5-folds, compared to the control. The purified tyrosinase was strongly inhibited by kojic acid and A. flavus DCM extracts with IC₅₀ values of 15.1 and 12.6 µg/mL, respectively. From the spectroscopic analysis, the main anti-tyrosinase compounds of A. flavus extract was resolved, and verified as undecanoic acid. Further studies are ongoing to unravel the in vivo effect and cytotoxicity of this compound in fungi and human, that could be a novel drug to various diseases associated with hyperpigmentation by melanin.     

A Comparative Study between Conventional and Advanced Extraction Techniques: Pharmaceutical and Cosmetic Properties of Plant Extracts

This study aimed to compare the influence of extraction methods on the pharmaceutical and cosmetic properties of medicinal and aromatic plants (MAPs). For this purpose, the dried plant materials were extracted using advanced (microwave (MAE), ultrasonic (UAE), and homogenizer (HAE) assisted extractions) and conventional techniques (maceration, percolation, decoction, infusion, and Soxhlet). The tyrosinase, elastase, α-amylase, butyryl, and acetylcholinesterase inhibition were tested by using L-3,4 dihydroxy-phenylalanine, N-Succinyl-Ala-Ala-p-nitroanilide, butyryl, and acetylcholine as respective substrates. Antioxidant activities were studied by ABTS, DPPH, and FRAP. In terms of extraction yield, advanced extraction techniques showed the highest values (MAE > UAE > HAE). Chemical profiles were dependent on the phenolic compounds tested, whereas the antioxidant activities were always higher, mainly in infusion and decoction as a conventional technique. In relation to the pharmaceutical and cosmetic properties, the highest inhibitory activities against α-amylase and acetylcholinesterase were observed for Soxhlet and macerated extracts, whereas the highest activity against tyrosinase was obtained with MAE > maceration > Soxhlet. Elastase and butyrylcholinesterase inhibitory activities were in the order of Soxhlet > maceration > percolation, with no activities recorded for the other tested methods. In conclusion, advanced methods afford an extract with high yield, while conventional methods might be an adequate approach for minimal changes in the biological properties of the extract.     

Valonea Tannin: Tyrosinase Inhibition Activity,Structural Elucidation and Insights into the Inhibition Mechanism

Valonea tannin is a natural product readily extracted from acorn shells that has been suggested to have potential skin whitening properties. This study investigated the tyrosinase inhibition activity of extracted valonea tannin and the associated structure–function activity. Nuclear magnetic resonance spectroscopy and molecular weight analysis with gel permeation chromatography revealed that valonea tannin could be characterized as a hydrolysable tannin with galloyl, hexahydroxydiphenoyl and open formed-glucose moieties and an average molecular weight of 3042 ± 15 Da. Tyrosinase inhibition assays demonstrated that valonea tannin was 334 times more effective than gallic acid and 3.4 times more effective than tannic acid, which may relate to the larger molecular size. Kinetic studies of the inhibition reactions indicated that valonea tannin provided tyrosinase inhibition through mixed competitive–uncompetitive way. Stern–Volmer fitted fluorescence quenching analysis, isothermal titration calorimetry analysis and in silico molecule docking showed valonea tannin non-selectively bound to the surface of tyrosinase via hydrogen bonds and hydrophobic interactions. Inductively coupled plasma-optical emission spectroscopy and free radical scavenging assays indicated the valonea tannin had copper ion chelating and antioxidant ability, which may also contribute to inhibition activity. These results demonstrated the structure–function activity of valonea tannin as a highly effective natural tyrosinase inhibitor that may have commercial application in dermatological medicines or cosmetic products.     

Quick screening of true tyrosinase inhibitors from natural products using tyrosinase‐immobilized magnetic nanoparticles and a magnetic microplate

Tyrosinase inhibitors from natural products have applications in the pharmaceutical, food, and cosmetic industries because of the functions of tyrosinase in skin disorders and in the enzymatic browning of fruits. Current in vitro inhibitor screening assays are based on the inhibition of the oxidation of l ‐3,4‐dihydroxyphenylalanine (l ‐DOPA) mediated by a mushroom tyrosinase. However, in these assays, a tyrosinase inhibitor or an antioxidant could inhibit dopaquinone formation. In this study, we aimed to eliminate this ambiguity by using a microplate assay integrating tyrosinase‐immobilized magnetic nanoparticles (TYR‐MNPs) and a homemade magnetic microplate for high‐throughput screening. After incubating extracts of natural products with TYR‐MNPs, the magnetic nanoparticles are attracted to the bottoms of wells, the extracts are rinsed, and TYR‐MNPs react with l ‐DOPA. This method can be used to screen compounds that interact with the active sites of the enzyme, or copper chelators that bind more strongly than tyrosinase to copper ions, distinguishing them from antioxidants or tyrosinase substrates. Integration with the homemade magnetic microplate enables high‐throughput inhibitor screening. Aloe vera flowers are crop by‐products, and litchi flowers fall after the blossom. Our work demonstrated that these flowers have tyrosinase inhibitory effects, thus increasing their value.     

Inhibitory effects of 1,3-selenazol-4-one derivatives on mushroom tyrosinase

This study reports depigmenting potency of 1,3-selenazol-4-one derivatives, which would be based upon the finding of direct inhibition to mushroom tyrosinase. 1,3-Selenazol-4-one derivatives exhibited inhibitory effect on dopa oxidase activity of mushroom tyrosinase. In this study, inhibitory effects of six kinds of 1,3-selenazol-4-one derivatives (A, B, C, D, E and F) on mushroom tyrosinase were investigated. Compounds at a concentration of 500 microM exhibited 33.4-62.1% of inhibition on dopa oxidase activity of mushroom tyrosinase. Their inhibitory effects were higher than that of kojic acid (31.7%), a well known tyrosinase inhibitor. 2-(4-Methylphenyl)-1,3-selenazol-4-one (A) exhibited the strongest inhibitory effect among them dose-dependently and in competitive inhibition manner.     

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.     

NMF-Based Approach for Missing Values Imputation of Mass Spectrometry Metabolomics Data

In mass spectrometry (MS)-based metabolomics, missing values (NAs) may be due to different causes, including sample heterogeneity, ion suppression, spectral overlap, inappropriate data processing, and instrumental errors. Although a number of methodologies have been applied to handle NAs, NA imputation remains a challenging problem. Here, we propose a non-negative matrix factorization (NMF)-based method for NA imputation in MS-based metabolomics data, which makes use of both global and local information of the data. The proposed method was compared with three commonly used methods: k-nearest neighbors (kNN), random forest (RF), and outlier-robust (ORI) missing values imputation. These methods were evaluated from the perspectives of accuracy of imputation, retrieval of data structures, and rank of imputation superiority. The experimental results showed that the NMF-based method is well-adapted to various cases of data missingness and the presence of outliers in MS-based metabolic profiles. It outperformed kNN and ORI and showed results comparable with the RF method. Furthermore, the NMF method is more robust and less susceptible to outliers as compared with the RF method. The proposed NMF-based scheme may serve as an alternative NA imputation method which may facilitate biological interpretations of metabolomics data.