Tyrosinase Assay Research Articles

Phytochemical Analysis and Assessment of Antioxidant and Enzyme Inhibitory Activity of Alchemilla pseudocartalinica Juz.

This study aimed to evaluate and compare the antioxidant capacity and enzyme inhibitory activity of extracts, sub-extracts, and fractions prepared from the aerial parts and roots of A. pseudocartalinica. The phytochemical content of the active extracts was also analyzed. According to the results, ellagic acid (38.42 mg/g) was the major compound in the aerial part methanol extract and catechin (185.30 mg/g) in the root methanol extract. The DPPH inhibition activity of all fractions was monitored, with the most active one (Fr B) reaching an IC50 value of (4.92±0.59 μg/mL). All the fractions prepared from the aerial parts' water sub-extract showed higher a-glucosidase inhibitory activity than the positive control acarbose. In the tyrosinase assay, Fr B (58.81±7.50 μg/mL) exhibited the highest inhibitory actions among all fractions. The structure of the major substances of the most active fraction were elucidated as quercetin 7-O-β-glucopyranosyl-3-O-β-glucuronopyranoside- (1), and α-[(2-formyl-5-hydroxymethyl)pyrrol-1-yl]aspartic acid (2).

Isolation and development of rosmarinic acid derivatives from Zostera marina L. and evaluating their hypopigmentation activity: In vitro and in silico study

Eelgrass, Zostera marina L., is vital to marine ecosystems, providing habitat, filtering pollutants, improving water quality, and serving as a source of bioactive compounds for pharmaceuticals. This study aimed to isolate and identify phenolic compounds from Z. marina, biotransform the main metabolite, rosmarinic acid (RA), and evaluate the anti-tyrosinase activity of these compounds. Various chromatographic techniques were employed to isolate compounds from Z. marina (1-4). Compound 1, exhibiting broad pharmaceutical activity, was biotransformed by Deinococcus geothermalis amylosucrase (DgAS) into two new glycosylated products (5-6). The isolated and biotransformed compounds were tested for anti-tyrosinase activity using a mushroom tyrosinase assay. Molecular docking and quantum chemical calculations were conducted to clarify the binding mode and reactivity of the compounds. The isolated compounds were identified via NMR and HR-MS as rosmarinic acid (RA) 1, methyl rosmarinate 2, methyl isorinate 3, and pinoresinol 4′-O-β-D-glucopyranoside 4. This is the first report of isolating and identifying compounds (2-4) from the Zosteraceae family. The glycosylated compounds were identified as rosmarinic acid 4-O-α-D-glucopyranoside 5 and rosmarinic acid 3′-O-α-D-glucopyranoside 6. Among all compounds, (4-6) showed significant tyrosinase inhibition, with rates of 55.5%, 22.3%, and 30.8%, respectively, compared to arbutin. The two glycosylated products exhibited better anti-tyrosinase activity than 1. In silico results aligned with in vitro findings. This study highlights Z. marina’s potential as a source of polyphenolic compounds with significant anti-aging activity, emphasizing the discovery of new anti-aging ingredients to enhance quality of life.

Discovery of (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide among N-substituted cinnamamide derivatives as a novel cosmetic ingredient for hyperpigmentation

Hyperpigmentation disorders may result from inappropriate melanin deposition and/or excessive melanin synthesis. They are classified mainly as aesthetic problems, but they can significantly affect human health by decreasing self-esteem. There are available only limited treatment options for hyperpigmentation disorder, among others, cosmetic products applied topically. Depigmenting ingredients were found to be ineffective and characterized by various side effects. As a result, many efforts are made to discover novel, potent, and safe melanogenesis inhibitors for possible use in topical cosmetic depigmenting formulations. Cinnamic acid derivatives constitute a widely tested group for that purpose. This article reports research in the group of N-alkyl cinnamamide derivatives (un)substituted in phenyl ring. Among tested series, (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide (compound 21) showed the most promising inhibitory properties in mushroom tyrosinase assay (IC50 = 36.98 ± 1.07 µM for monophenolase activity, IC50 = 146.71 ± 16.82 µM for diphenolase activity) and melanin production inhibition in B16F10 mouse melanoma cell line at concentration 6.25 µM resulting probably from decreasing of Tyr, Mitf, Tyrp-1, and Tyrp-2 genes expression. This compound also showed melanin production inhibitory properties in pigmented reconstructed human epidermis when used in 1 % and 2 % solutions in 50 % PEG400. In vitro evaluation of its safety profile showed no cytotoxicity to human keratinocytes HaCaT, human skin fibroblasts BJ, and human primary epidermal melanocytes HEMa, no mutagenicity in the Ames test, no genotoxicity in micronucleus test, no phototoxicity, as well as no skin irritation potential tested in PEG400 solution. This compound was also shown to penetrate across the epidermis to reach the possible site of action. The performed research led to classify (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide as a novel potential depigmenting cosmetic ingredient.

ANALYSIS OF VOLATILE COMPOUNDS OF ALCEA CALVERTII BOISS. AND ITS ANTIMICROBIAL, ANTICHOLINESTERASE, AND ANTITYROSINASE POTENCY

The research aimed to analyze the volatile compounds by using SPME-GC-MS and to detect anticholinesterase, antityrosinase, and antimicrobial potential of metanol extract of Alceacalvertii Boiss., an endemic species for Türkiye. The inhibitory effects of tyrosinase, acetylcholinesterase, and butyrylcholinesterase of the plant were determined by spectroscopic technique and the plant’s antimicrobial activity was assessed using the agar diffusion method. A total of 18 volatile compounds were specified belonging to terpenes classes. o-cymene (10.60%) and sesquicineole (15.55%) were detected as major volatile components of the species by the SPME-GC-MS technique. Meaningful antimicrobial activity was observed on Candida tropicalis, Enterococcus faecalis, Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus, and Pseudomonas aeruginosa. The half maximal inhibitory concentration (IC50) value of the plant was determined 60.12 ± 1.75 μg/mL as a result of tyrosinase assay. IC50 value was found 111.54 ± 1.75 μg/mL, according to butyrylcholinesterase inhibition studies. Considering all the findings, it has been determined that the plant includes diverse volatile compounds and showed promising antimicrobial, tyrosinase inhibitory, and moderate butyrylcholinesterase inhibitory effects so Alceacalvertii may be the up-and-coming source of natural medicine for microbial and dermatological diseases although limited to its effects for Alzheimer's disease.

Elucidating anti-aging and antioxidant activity of Hydnocarpus anthelmintica on skin aging and cosmeceutical potentials: in silico and in vitro study.

Hydnocarpus anthelmintica (HA) has been traditionally used for treating leprosy and is known for its antioxidant and anti-inflammatory activities. The aim of this study was to investigate the active compounds and targets of HA extracts, involved in oxidative stress and skin aging. The active compounds and targets of HA extracts were identified using network pharmacology. The pathway study was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. HA semen was measured for its in-vitro antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and anti-aging activities using collagenase, elastase, and tyrosinase assays. A total of 21 intersecting core targets were identified from 8 compounds, 51 action targets, and 1810 skin aging and oxidative stress-associated target genes. A compound-target network was constructed, and 3 compounds (luteolin, beta-carotene and genkwanin), and 4 hub genes (TP53, HSP90AA1, JUN, and MAPK1) were identified. The KEGG pathway study revealed that the compounds were correlated with PI3K-Akt, p53, HIF-1, and MAPK signaling. The results of in-vitro experiments showed the effect of HA extract on oxidative stress reduction and collagenase inhibition. We discovered two main active compounds, luteolin and β-carotene, that may be involved in p53 and MAPK signaling, and showed HA extract activity against oxidative stress and collagenase.

Biological activities study of Cinnamon (Cinnamomum burmanni (Nees & T.Nees) Blume) as Cosmetic Ingredients

Objectives: The goals of this research were to study the biological activities of cinnamon bark extract related to its use as cosmetic ingredients. The potential use of cinnamon bark as cosmetic preparations was studied by confirming its antioxidative, antimicrobial, and its inhibition towards tyrosinase activity. Methods: Extraction was performed by maceration using 70% ethanol. Antioxidative activity using the DPPH methods, melanin inhibitory effect was determined using in vitro anti tyrosinase assay. MHC and MIC of cinnamon bark extract were determined towards Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes. Results: The standardization result of the crude drugs showed positive result on qualitative analysis of Flavonoid, Tannin, and Quinone groups. Flavonoid content also found on the ethanolic extract. The IC­50 antioxidative activity of cinnamon ethanolic was 7.52 ± 0.13 μg/mL compared to ascorbic acid with 2.54 + 0.02 μg/mL. The Ethanolic extract of cinnamon inhibited tyrosinase with IC­50 of 9.44 ± 0.26 mg/mL. Lowest minimum inhibitory concentration of ethanolic extract was found towards Cutibacterium acne with 1000 μg/mL. Whereas weak antimicrobial activity was showed towards Staphylococcus aureus and Staphylococcus epidermidis with MIC and MBC value started from 2000 μg/mL. Conclusion: Comparable result on antioxidative and anti-tyrosinase analysis shows that cinnamon bark have fair potential to further developed and formulated for topical maintenance product.

Ultrasound-assisted pumpkin tendril extracts inhibits melanogenesis by suppressing the CREB/MITF signaling pathway in B16F10 melanoma cells, zebrafish, and a human skin model

Agricultural byproducts are intermediate products generated during food processing and contain functional ingredients that are suitable for use in the green circular economy. This study aimed to demonstrate the skin-whitening effect of pumpkin tendril extract (PTE) using a mushroom tyrosinase assay, L-DOPA staining, qRT-PCR, western blotting, a zebrafish model, and a reconstituted 3D human skin model. PTE dose-dependently scavenged DPPH free radicals and inhibited melanogenesis stimulated by α-MSH without exhibiting cytotoxicity in 2D- and 3D-cultured B16F10 cells. Interestingly, PTE significantly reduced melanogenesis in zebrafish and 3D-pigmented human skin models. In addition, PTE notably downregulated the expression of proteins related to melanogenesis, such as MITF, TRP-1, TRP-2, and tyrosinase. Phosphorylation of CREB, an upregulator of MITF, was also attenuated by PTE treatment. Component analysis revealed that rutin is an active compound in PTE affecting melanogenesis. These results suggest that PTE suppresses melanogenesis and tyrosinase activity by regulating the CREB/MITF signaling pathway.

Bioactivity profiles of six baobab fruit pulp powders via planar chromatography hyphenated with effect-directed analysis

Baobab (Adansonia digitata) fruit pulp has a high nutrient content and has been traditionally used for medicinal purposes (e.g., as an anti-inflammatory and antioxidant agent) that may help protect against chronic diseases. Six different baobab fruit pulp powders were investigated using three different extractants and analyzed by high-performance thin-layer chromatography (HPTLC) hyphenated with antibacterial bioassays and enzyme inhibition assays. The developed non-target effect-directed screening was performed after extraction with pentyl acetate – ethanol 1:1 (V/V) on the HPTLC plate silica gel 60 using toluene – ethyl acetate – methanol 6:3:1 (V/V/V) as mobile phase system and derivatization via the anisaldehyde sulfuric acid reagent for detection. The physico-chemical profiles of the six baobab fruit pulp powder extracts were comparable, although the intensity of some zones was moderately different. The following effect-directed profiling via tyrosinase, α-glucosidase, and acetylcholinesterase inhibition assays as well as antibacterial Aliivibrio fischeri and Bacillus subtilis bioassays revealed one prominent multipotent bioactive compound zone in common, more or less active in all five studied (bio)assays. Via the recording of high-resolution mass spectra, this compound zone was tentatively assigned to coeluting saturated (palmitic acid 16:0 and stearic acid 18:0), monounsaturated (oleic acid 18:1), and polyunsaturated (linoleic acid 18:2 and linolenic acid 18:3) fatty acids. This finding was confirmed by other studies, which already proved individual activities of fatty acids. The first (bio)activity profiling of baobab fruit pulp powders via HPTLC-effect-directed analysis revealed that the baobab fruit could be considered as a functional food, however, further research is needed to study the impact on health and the influences on the bioactivity arising from different climates, years and soils or regions.

Fluorometric Assay of Tyrosinase and Atrazine Based on the Use of Carbon Dots and the Inhibition of Tyrosinase Activity.

Sensitive and convenient strategy of tyrosinase (TYR) and its inhibitor atrazine is in pressing demand for essential research as well as pragmatic application. In this work, an exquisite label-free fluorometric assay with high sensitivity, convenience and efficiency was described for detecting TYR and the herbicide atrazine on the basis of fluorescent nitrogen-doped carbon dots (CDs). The CDs were prepared via one-pot hydrothermal reaction starting from citric acid and diethylenetriamine. TYR catalyzed the oxidation of dopamine to dopaquinone derivative which could quench the fluorescence of CDs through a fluorescence resonance energy transfer (FRET) process. Thus, a sensitive and selective quantitative evaluation of TYR can be constructed on the basis of the relationship between the fluorescence of CDs and TYR activity. Atrazine, a typical inhibitor of TYR, inhibited the catalytic activity of TYR, leading to the reduced dopaquinone and the fluorescence was retained. The strategy covered a broad linear range of 0.1-150 U/mL and 4.0-80.0 nM for TYR and atrazine respectively with a low detection limit of 0.02 U/mL and 2.4 nM/mL. It is also demonstrated that the assay can be applied to detect TYR and atrazine in spiked complex real samples, which provides infinite potential in application of disease monitoring along with environmental analysis.

Anti-tyrosinase and antimelanogenic effect of cinnamic acid derivatives from Prunus mahaleb L.: Phenolic composition, isolation, identification and inhibitory activity.

The traditional use of Prunus species against skin diseases and especially for skin lightning cosmeceutical purposes is widespread in many cultures. Prunus mahaleb L. is a well known food plant and used in the baking industry for flavoring. The fruit kernels (endocarp) are used in India for hyperpigmentation. To investigate the chemical composition with the antimelanogenesis effect of P. mahaleb seed and kernel extracts and isolated compounds. Isolation studies performed from the methanol extracts obtained from kernels and structures were determined using NMR and MS analysis. Antimelanogenesis effect was determined by mushroom tyrosinase assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells. Five cinnamic acid derivatives were isolated and their structures (2-O-β-glucopyranosyloxy-4-methoxy-hydrocinnamic acid (1), cis-melilotoside (2), dihydromelilotoside (3), trans-melilotoside (4), 2-O-β-glucosyloxy-4-methoxy trans-cinnamic acid (5)) were elucidated using advanced spectroscopic methods. Mushroom tyrosinase enzyme inhibition of extracts, fractions and pure compounds obtained from P. mahaleb kernels were investigated and structure-activity relationship revealed. According to a detailed, comprehensive and validated LC-MS/MS technique analysis, vanilic acid (41.407mg/g), protocatechuic acid (8.992mg/g) and ferulic acid (4.962mg/g) in the kernel ethylacetate fraction; quinic acid (14.183mg/g), fumaric acid (8.349mg/g) and aconitic acid (5.574mg/g) were found as major phenolic compounds in the water fraction. The correlation of trace element copper content in extracts and fractions with mushroom enzyme activity was determined. By examining the enzyme kinetics of the compounds with effective cinnamic acid derivatives, inhibition types and enzyme binding constants Ki were calculated. Compounds 1,3 and 5 exhibited high noncompetitive tyrosinase inhibitory activity against L-tyrosine substrates, with IC50 values of 0.22, 0.31 and 0.37mM respectively. In addition compounds 1, 3 and 5 showed dose-dependent inhibitory effects on intracellular tyrosinase and melanin levels in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 melanoma cells. Potent tyrosinase inhibitory compounds and extracts of P. mahaleb kernels suggest that it could be a new, non-toxic and inexpensive resource for the cosmeceutical industry and in skin diseases associated with hyperpigmentation.

Antioxidant and Anti-tyrosinase Activities of Halymenia durvillei Water Extract Containing R-Phycoerythrin Before and After Microencapsulation

Halymenia durvillei water extract (HDWE) contains a dark-pink colored pigment of Rphycoerythrin (R-PE), which its stability is influenced by temperature and pH andpossesses biological activities. Microencapsulation could be the solution to preservingthe nature of R-PE. This study characterized the HDWE emulsion and analyzed the RPE content, antioxidants, and anti-tyrosinase activities before and after microencapsulation using sodium caseinate and maltodextrin (M-HDWE). Halymenia durvillei was extracted in distilled water (ratio 1:2, w/v) for 24 hours at 4 °C. Sodium caseinate to maltodextrin coating ratios were 1:2; 1:4; 1:6 and 1:12. Emulsion (viscosity, color, and R-PE content) and powder characteristics (surface morphology) were determined using Brookfield DV2T, ColorFlex EZ Hunterlab, UV-VIS spectrophotometry, and JeolSEM, respectively. The antioxidant and anti-tyrosinase activities were determined using the Ferric Reducing Antioxidant Power (FRAP) and mushroom tyrosinase assays. The Commision Internationale d’Éclairage (CIE) L* a* b* colorimetry result showed that the more maltodextrin and the lower viscosity, the more intense the emulsion color. Despite HDWE having roughly six-fold more R-PE content, all M-HDWE treatment samples exhibited equal R-PE contents (p 0.05). The M-HDWE 1:2; 1:4; 1:6; and 1:12 microcapsules had more wrinkled spheres and fewer cracks than MHDWE 0. The M-HDWE 1:2 had 33.10% lower antioxidant activity than the HDWE and was the highest compared to other M-HDWE compositions (17.15±1.27 µM/µg extract, p 0.05). Meanwhile, all M-HDWE had low anti-tyrosinase activity. It can be concluded that microencapsulation could be a solution for preserving HDWE’s antioxidant activity but not its anti-tyrosinase activity

Antioxidant and Anti-tyrosinase Activities of Halymenia durvillei Water Extract Containing R-Phycoerythrin Before and After Microencapsulation

Halymenia durvillei water extract (HDWE) contains a dark-pink colored pigment of Rphycoerythrin (R-PE), which its stability is influenced by temperature and pH and possesses biological activities. Microencapsulation could be the solution to preserving the nature of R-PE. This study characterized the HDWE emulsion and analyzed the RPE content, antioxidants, and anti-tyrosinase activities before and after microencapsulation using sodium caseinate and maltodextrin (M-HDWE). Halymenia durvillei was extracted in distilled water (ratio 1:2, w/v) for 24 hours at 4 °C. Sodium caseinate to maltodextrin coating ratios were 1:2; 1:4; 1:6 and 1:12. Emulsion (viscosity, color, and R-PE content) and powder characteristics (surface morphology) were determined using Brookfield DV2T, ColorFlex EZ Hunterlab, UV-VIS spectrophotometry, and JeolSEM, respectively. The antioxidant and anti-tyrosinase activities were determined using the Ferric Reducing Antioxidant Power (FRAP) and mushroom tyrosinase assays. The Commision Internationale d’Éclairage (CIE) L* a* b* colorimetry result showed that the more maltodextrin and the lower viscosity, the more intense the emulsion color. Despite HDWE having roughly six-fold more R-PE content, all M-HDWE treatment samples exhibited equal R-PE contents (p 0.05). The M-HDWE 1:2; 1:4; 1:6; and 1:12 microcapsules had more wrinkled spheres and fewer cracks than MHDWE 0. The M-HDWE 1:2 had 33.10% lower antioxidant activity than the HDWE and was the highest compared to other M-HDWE compositions (17.15±1.27 µM/µg extract, p 0.05). Meanwhile, all M-HDWE had low anti-tyrosinase activity. It can be concluded that microencapsulation could be a solution for preserving HDWE’s antioxidant activity but not its anti-tyrosinase activity

Effect of Aspergilus oryzae-fermented broken rice, brewers’ rice and rice bran on melanogenesis in highly pigmented human melanoma, MNT-1

The food and beauty sectors are developing strategies to establish a link between nutrient consumption and skin health, as nutraceuticals offer a promising treatment option for some skin disorders such as hyperpigmentation and premature ageing. As a result, the consumption of food ingredients and supplements claiming to lower the risk of developing such skin problems is increasing. In this study, anti-tyrosinase potential of Aspergillus oryzae-fermented rice by-product water extracts namely fermented broken rice (FBR), brewers’ rice (FBrR), and rice bran (FRB) was assessed through in vitro study using highly pigmented human melanoma MNT-1 cells. The fermented extracts were evaluated for cytotoxicity, anti-tyrosinase activity using intracellular tyrosinase assay as well as melanin content analysis. Their effect on the gene expression of three melanogenic enzymes: tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein 2 (TRP-2) was also assessed by qPCR analysis. As a result, all extracts at tested concentrations of 50 and 100 µg/mL have a low cytotoxicity effect. Together with the positive control kojic acid, the FBR at both concentrations and the FRB at 50 µg/mL significantly decreased intracellular tyrosinase activity. However, their melanin content was not significantly reduced. The qPCR analysis indicated that FBR at 50 and 100 µg/ mL significantly reduced TYR gene expression by up to 71% and 61%, respectively. On the other hand, FBR (100 µg/mL) increased TRP-1 gene expression, whilst FRB (100 µg/ mL) elevated both TRP-1 and TRP-2 gene expression. The positive control kojic acid significantly reduced TYR, TRP-1 and TRP-2 gene expression. However, FBrR demonstrated no significant effect in any of the analyses. Based on these findings, FBR and FRB at 50 µg/mL possess anti-tyrosinase potential. However, further investigation is needed to dissect deeper mechanisms underlying their potential as functional bioingredients for nutraceutical or cosmeceutical applications.

Anti-Melanogenic and Anti-Oxidative Effects of Nostoc verrucosum (ashitsuki) Extracts

Nostoc verrucosum, an edible microalgal species, forms colonies in streams. Here, we investigated the antimelanogenic and anti-oxidative effects of N. verrucosum extracts. We collected N. verrucosum from Toyama Prefecture, Japan, and successfully cultured it in indoor cultivation systems. Aqueous, methanol, and hexane extracts of N. verrucosum were prepared for various experiments. To elucidate the antimelanogenic effects of N. verrucosum, we performed tyrosinase assay, melanin content assay, western blotting, and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Anti-oxidative effects were evaluated using the 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay. The methanol and hexane extracts significantly inhibited melanin synthesis in B16F1 melanoma cells. Western blotting showed that 12.5 and 25.0 µg/mL N. verrucosum hexane extract suppressed tyrosinase activity. The qRT-PCR analysis revealed that N. verrucosum hexane extract inhibited α-melanocyte stimulating hormone-enhanced tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and melanocortin 1 receptor (MC1R) mRNA expression. On the contrary, N. verrucosum hexane extract did not alter microphthalmia-associated transcription factor (Mitf) transcription. The ABTS assay showed that all extracts had radical scavenging activities, and the IC50 values of the aqueous and methanol extracts were 294.6 and 172.8 µg/mL, respectively. Our findings demonstrate that N. verrucosum is a suitable candidate for the development of antimelanogenic agents, cosmetics, or functional food ingredients.

Computer-Aided Virtual Screening and In Vitro Validation of Biomimetic Tyrosinase Inhibitory Peptides from Abalone Peptidome.

Hyperpigmentation is a medical and cosmetic problem caused by an excess accumulation of melanin or the overexpression of the enzyme tyrosinase, leading to several skin disorders, i.e., freckles, melasma, and skin cancer. Tyrosinase is a key enzyme in melanogenesis and thus a target for reducing melanin production. Although abalone is a good source of bioactive peptides that have been used for several properties including depigmentation, the available information on the anti-tyrosinase property of abalone peptides remains insufficient. This study investigated the anti-tyrosinase properties of Haliotis diversicolor tyrosinase inhibitory peptides (hdTIPs) based on mushroom tyrosinase, cellular tyrosinase, and melanin content assays. The binding conformation between peptides and tyrosinase was also examined by molecular docking and dynamics study. KNN1 showed a high potent inhibitory effect on mushroom tyrosinase with an IC50 of 70.83 μM. Moreover, our selected hdTIPs could inhibit melanin production through the reductions in tyrosinase activity and reactive oxygen species (ROS) levels by enhancing the antioxidative enzymes. RF1 showed the highest activity on both cellular tyrosinase inhibition and ROS reduction. leading to the lower melanin content in B16F10 murine melanoma cells. Accordingly, it can be assumed that our selected peptides exhibited high potential in medical cosmetology applications.

Influence of pyrrolizidine alkaloids depletion upon the biological activity of Symphytum officinale L. extracts

Ethnopharmacological relevanceComfrey (Symphytum officinale L., Boraginaceae) root preparations are used as both traditional remedies and therapeutic agents in treating pain and inflammation associated with joint, bone, and muscle ailments. Even though numerous phytochemicals contribute to the beneficial effects of comfrey, the presence of toxic pyrrolizidine alkaloids (PAs) overshadows its uses. Aim of the studyIn this work, different PA-/mucilage-depleted/undepleted comfrey root extracts were subjected to detailed phytochemical characterization and biological evaluation. Materials and methodsThe phytochemical profiling was performed by LC-HRMS/MS. The quantification of PAs and major phenolic compounds was carried out by LC-MS/MS and LC-DAD. Antioxidant and enzyme inhibitory activity was determined by in vitro free radical scavenging, ion reducing, metal chelating, cholinesterase, tyrosinase, amylase, and glucosidase assays. Using an ex vivo model of LPS-stimulated neutrophils, their viability (as measured by flow cytometry) and the release of IL-1β, IL-8, and TNF-α were determined (ELISA assay). Results12 phenolic acids, six PAs, three organic acids, two fatty acids, and two sugars were identified in the obtained comfrey extracts. The PA-depleted materials contained PAs levels below 2 ppm, whereas the removal of mucilage increased the content of rosmarinic acid, globoidnan A, globoidnan B, and rabdosiin. PA-depletion did not significantly affect the antioxidant potential. However, the radical scavenging and metal reducing properties were higher in the mucilage-depleted extracts. Neither PA-depletion nor mucilage-depletion had considerable effects on the in vitro inhibitory activity of cholinesterases, tyrosinase, amylase, and glucosidase or release of ex vivo pro-inflammatory cytokines (e.g., IL-1β, IL-8, and TNF-α) in LPS-stimulated neutrophils. ConclusionsIn light of their superior safety profiles, PA-depleted comfrey extracts can be utilized further in cosmetic and pharmaceutical products.

A Blue/NIR ratiometric fluorescent probe for intracellular detection of Tyrosinase and the inhibitor screening

A novel ratiometric fluorescent tyrosinase assay is developed based on hybrid nano-assembly of gold nanocluster and tyrosine-containing peptides. The AuNCs@YCY nano-probe (AYNP) is fabricated through the hydrophobic interactions and π-π stacking between the tyrosine residues of the Tyr-Cys-Tyr tripeptide (YCY) and the ligands on the surfaces of AuNCs under the near-isoelectric pH value. The resulted AYNP shows distinct fluorescence responses, spontaneous turn-on of the blue emission and turn-off of the near-infrared emission, with a single wavelength excitation. It is demonstrated that the enhancement and quenching are due to the production of pheomelanin and dopaquinone structures, respectively, induced by tyrosinase oxidation. The internal referencing system provides the tyrosinase assay with superior sensitivity and a detection limit as low as 6.3 U L−1 could be achieved. The experimental results also demonstrate the excellent selectivity, good photo-stability, and both in vitro and cellular applications of AYNP. This assay technique is low-cost, easy to prepare, and shows excellent potential as a novel melanoma clinical diagnostic platform and a tyrosinase inhibitor screening tool.

Anserine/Carnosine-Rich Extract from Thai Native Chicken Suppresses Melanogenesis via Activation of ERK Signaling Pathway.

Skin hyperpigmentation is an aesthetic problem that leads to psychosocial issues. Thus, skin whitening agents from agro- and poultry-industrial co-products are considered high economic value ingredients of interest for sustainable application. Therefore, this study aimed to determine the cosmeceutical potential of anserine/carnosine-rich chicken extract (ACCE) from the Thai native chicken Pradu Hang Dam Mor Kor 55 (PD) meat. The chemical composition was identified and quantified using the HPLC-UV method. Then, the antioxidation potential of the extract was compared to that of L-anserine and L-carnosine, using 1,1-diphenyl-2-picrylhydrazyl assay and shikonin-induced production of reactive oxygen species in CCD-986Sk cell models, and the anti-melanogenesis effect in the MNT-1 melanoma cell line model was investigated. Furthermore, related mechanisms were identified using colorimetric tyrosinase assay and the Western blot technique. The ACCE was composed of L-anserine and L-carnosine as two major constituents. In a dose-dependent manner, ACCE, L-anserine, and L-carnosine manifested significant antioxidation potential and significant reduction of melanin production. Activation of the extracellular signal-regulated kinase (ERK) signaling pathway and inhibition of tyrosinase activity of ACCE were demonstrated as the mechanisms of the anti-melanogenesis effect. In conclusion, ACCE has been revealed as a potential cosmeceutical agent due to its antioxidation and anti-melanogenic activity in association with L-anserine and L-carnosine composition and biomolecular regulating ability. Therefore, further studies and development should be considered to support the utilization of anserine/carnosine-rich chicken extract in the cosmetic industry for economic value creation and sustainability.

Evaluating the phyto-complexity and poly-pharmacology of spices: The case of Aframomum melegueta K. Schum (Zingiberaceae)

The seeds of Aframomum melegueta K. Schum (grains of paradise, Zingiberaceae) are used as a common spice in African countries and a fine condiment in the European cuisine. In this study, we evaluated the phytochemical profile of various seed extracts of A. melegueta as well as their antimicrobial, antioxidant and enzyme inhibitory effects. In total, 25 diarylheptanoids, five gingerol derivatives and nine phenolic/organic acids were tentatively annotated in A. melegueta by liquid chromatography hyphenated with high resolution tandem mass spectrometry (LC-HRMS/MS). A. melegueta showed a moderate inhibitory activity against different human pathogenic microbial strains, with H. pylori the most sensitive microorganism. A strong antioxidant and enzyme inhibitory potential was shown in various radical scavenging, reducing and chelating assays as well as in cholinesterase, tyrosinase and glucosidase assays. Several specialized metabolites from A. melegueta (diarylheptanoids, gingerols) were shown to be directly linked with the investigated antioxidant and enzyme inhibitory activities, as evaluated through the correlation analysis. In addition, two diarylheptanoids (one heptan-3-ol and one heptan-3,5-diol) displayed the strongest binding to acetylcholinesterase (AChE) via multiple H-bonds, a couple of π-π interactions and van der Waals interactions all over the catalytic channel of the enzyme, as evidenced by the molecular docking study. Overall, our work brings new contributions to the phyto-complexity and poly-pharmacology of spices from genus Aframomum than can find future applications in pharmaceutical, nutraceutical or cosmeceutical industry.

Mushroom Tyrosinase Assay is used to Teach Enzyme Kinetics and Inhibition Studies in an Undergraduate Biochemistry Course

A spectrophotometric mushroom tyrosinase assay was optimized for use in the undergraduate biochemistry laboratory course at Virginia Tech. The laboratory exercise is designed to teach students about enzyme kinetics and tyrosinase‐inhibitor interactions. The tyrosinase protein structure was first studied using the python based molecular visualization software PyMOL and a PDB ID from the Protein Data Bank. The PDB ID that was used was PY9W and displays a H(2) L(2) tetramer. The H subunits of the complex contain a copper binding site that is coordinated by 3 histidine residues. The structure/function relationship was further studied using an immersive modeling hypercube. The assay uses white button mushrooms as the source of the tyrosinase enzyme, l‐3,4 dihydroxyphenylalanine (L‐DOPA) as the substrate, and kojic acid as the inhibitor. The optimal amounts of enzyme, substrate, and inhibitor are first determined, then a series of experiments are conducted which test varying concentrations of L‐DOPA against varying concentrations of kojic acid. Change in absorbance is measured at 475 nm in a spectrophotometer and the data is plotted to determine the kinetic constants and type of inhibition. Experimental kinetic constants are then compared to previously reported values. Further protein concentration in the tyrosinase extract is determined using Bradford assays. The incorporation of this exercise into the undergraduate biochemistry laboratory course at Virginia Tech will allow students to explore enzyme kinetics and tyrosinase‐inhibitor interactions.