6-Methylcoumarin Promotes Melanogenesis through the PKA/CREB, MAPK, AKT/PI3K, and GSK3ß/ß-Catenin Signaling Pathways.

We investigated the effects of four coumarin derivatives, namely, 6-methylcoumarin, 7-methylcoumarin, 4-hydroxy-6-methylcoumarin, and 4-hydroxy-7-methylcoumarin, which have similar structures on melanogenesis in a murine melanoma cell line from a C57BL/6J mouse called B16F10. Our results showed that only 6-methylcoumarin significantly increased the melanin synthesis in a concentration-dependent manner. In addition, the tyrosinase, TRP-1, TRP-2, and MITF protein levels were found to significantly increase in response to 6-methylcoumarin in a concentration-dependent manner. To elucidate the molecular mechanism whereby 6-methylcoumarin-induced melanogenesis influences the melanogenesis-related protein expression and melanogenesis-regulating protein activation, we further assessed the B16F10 cells. The inhibition of the ERK, Akt, and CREB phosphorylation, and conversely, the increased p38, JNK, and PKA phosphorylation activated the melanin synthesis via MITF upregulation, which ultimately led to increased melanin synthesis. Accordingly, 6-methylcoumarin increased the p38, JNK, and PKA phosphorylation in the B16F10 cells, whereas it decreased the phosphorylated ERK, Akt, and CREB expressions. In addition, the 6-methylcoumarin activated GSK3ß and ß-catenin phosphorylation and reduced the ß-catenin protein level. These results suggest that 6-methylcoumarin stimulates melanogenesis through the GSK3ß/ß-catenin signal pathway, thereby affecting the pigmentation process. Finally, we tested the safety of 6-methylcoumarin for topical applications using a primary human skin irritation test on the normal skin of 31 healthy volunteers. We found that 6-methylcoumarin did not cause any adverse effects at concentrations of 125 and 250 µM. Our findings indicate that 6-methylcoumarin may be an effective pigmentation stimulator for use in cosmetics and the medical treatment of photoprotection and hypopigmentation disorders.

6-Methylcoumarin rescues bacterial quorum sensing induced ribosome-inactivating stress in Caenorhabditis elegans.

INTRODUCTION: Bacterial pathogenicity has for long posed severe effects on patient care. Pseudomonas aeruginosa is a common cause of hospital-acquired infections and nosocomial illnesses. It is known to infect the host by colonizing through quorum sensing and the production of exotoxins. METHODS: The current effort is an analysis of proteomic alterations caused by P. aeruginosa PAO1 to study the effects of quorum sensing inhibitor 6-Methylcoumarin on PAO1 infectivity in the Caenorhabditis elegans model. RESULTS: Through tandem mass tag-based quantitative proteomics approaches, 229 proteins were found to be differentially regulated in infection and upon inhibition. Among these, 34 proteins were found to be dysregulated in both infection and quorum-sensing inhibition conditions. Along with the dysregulation of proteins involved in host-pathogen interaction, PAO1 was found to induce ribosome-inactivating stress accompanied by the downregulating mitochondrial proteins. This in turn caused dysregulation of apoptosis. The expression of multiple proteins involved in ribosome biogenesis and structure, oxidative phosphorylation, and mitochondrial enzymes were altered due to infection. This mechanism, adapted by PAO1 to survive in the host, was inhibited by 6-Methylcoumarin by rescuing the downregulation of ribosomal and mitochondrial proteins. CONCLUSIONS: Taken together, the data reflect the molecular alterations due to quorum sensing and the usefulness of inhibitors in controlling pathogenesis.

Discovery of a novel plant-derived agent against Ralstonia solanacearum by targeting the bacterial division protein FtsZ.

Ralstonia solanacearum (R. solanacearum) is one of the most devastating bacterial pathogens and leads to serious economic losses in crops worldwide. In this study, the antibacterial activities of novel plant-derived coumarins against R. solanacearum and their underlying mechanisms were initially investigated. The bioactivity assay results showed that certain coumarins had significant in vitro inhibitory effects against R. solanacearum. Notably, 6-methylcoumarin showed the best in vitro antibacterial activity with 76.79%. Interestingly, 6-methylcoumarin was found to cause cell elongation, disrupt cell division, and suppress the expression of the bacterial division protein coding genes ftsZ. Compared with the control treatment, the ∆ftsZ mutant inhibited bacterial growth and caused the bacteria to be more sensitive to 6-methylcoumarin. The application of 6-methylcoumarin effectively suppressed the development of tobacco bacterial wilt in pot and field experiments, and significantly reduced the bacterial population in tobacco stems. The control efficiency of 6-methylcoumarin treatment was 35.76%, 40.51%, 38.99% at 10, 11, and 12 weeks after tobacco transplantation in field condition. All of these results demonstrate that 6-methylcoumarin has potential as an eco-friendly and target specificity agent for controlling tobacco bacterial wilt.

Anti-Inflammatory Effects of 6-Methylcoumarin in LPS-Stimulated RAW 264.7 Macrophages via Regulation of MAPK and NF-κB Signaling Pathways.

Persistent inflammatory reactions promote mucosal damage and cause dysfunction, such as pain, swelling, seizures, and fever. Therefore, in this study, in order to explore the anti-inflammatory effect of 6-methylcoumarin (6-MC) and suggest its availability, macrophages were stimulated with lipopolysaccharide (LPS) to conduct an in vitro experiment. The effects of 6-MC on the production and levels of pro-inflammatory cytokines (interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α) and inflammatory mediators (nitric oxide (NO), prostaglandin E2 (PGE2)) in LPS-stimulated RAW 264.7 cells were examined. The results showed that 6-MC reduced the levels of NO and PGE2 without being cytotoxic. In addition, it was demonstrated that the increase in the expression of pro-inflammatory cytokines caused by LPS stimulation, was decreased in a concentration-dependent manner with 6-MC treatment. Moreover, Western blot results showed that the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which increased with LPS treatment, were decreased by 6-MC treatment. Mechanistic studies revealed that 6-MC reduced the phosphorylation of the mitogen-activated protein kinase (MAPK) family and IκBα in the MAPK and nuclear factor-kappa B (NF-κB) pathways, respectively. These results suggest that 6-MC is a potential therapeutic agent for inflammatory diseases that inhibits inflammation via the MAPK and NF-κB pathways.

Biodistribution study of free and microencapsulated 6-methylcoumarin in Wistar rats by HPLC.

A sensitive, specific and reproducible HPLC method has been developed and validated for the quantitative determination of 6-methylcoumarin (6MC) in plasma and other tissues in Wistar rats. A C18 column was used with UV detection at 321 nm and a gradient system consisting of methanol-deionized water was used as mobile phase. The retention time for 6MC was 14.921 min and no interfering peaks were observed for any of the matrices. Linear relationships (r(2) > 0.997) were obtained between the peak height ratios and the corresponding biological sample concentrations over the range 0.4-12.8 µg/mL. Precision and accuracy were evaluated; the coefficient of variation and the relative error for all of the organs were <2 and 7%, respectively. The limit of quantitation was 0.20 µg/mL for the heart and 0.30 µg/mL for the other tissues evaluated. This HPLC method was successfully used in the determination of 6MC in the biodistribution study after administration of 200 mg/kg of both 6MC-free and 6MC-loaded polymeric microparticles. In this study, extensive 6MC was found, in both free and microencapsulated forms, in all the organs tested. The 6MC-free showed a range of between 1.7 and 11.5 µg/g, while the microencapsulated 6MC showed concentrations of between 6.35 and 17.7 µg/g, suggesting that 6MC improved absorption rate.

6-methylcoumarin/miR-122 suppresses hepatic Sortilin-mediated ApoB-100 secretion to attenuate aortic atherosclerosis.

This study aimed to investigate the effects of hepatic microRNA-122 (miR-122) on Sortilin-mediated apolipoprotein B100 (apoB-100) secretion, and on aortic lipid deposition and atherosclerosis (AS) lesions and to clarify the antiatherosclerotic mechanism of 6-methylcoumarin (6-MC) via the modulation of miR-122. Bioinformatics analysis revealed that miR-122 was putatively overexpressed in a liver-specific manner and was downregulated in steatotic livers. miR-122 was shown to suppress the expression of Sortilin by complementarily pairing to the 3'-untranslated region (3'-UTR) of Sortilin mRNA via bioinformatics and dual-luciferase reporter assays, impeding Sortilin-mediated apoB-100 secretion from HepG2 cells. Administration of 6-MC significantly upregulated hepatocellular miR-122 levels, reducing Sortilin expression and apoB-100 secretion in HepG2 cells. The miR-122 mimic vigorously enhanced 6-MC-depressed Sortilin expression, while miR-122 inhibitor repealed the inhibitory effect of 6-MC on Sortilin expression to some extent in HepG2 cells. After internal intervention with the miR-122 precursor, and 6-MC supplementation alone or in combination with the miR-122 sponge led to the reduction in blood triglyceride (TG) levels, low-density lipoprotein-cholesterol (LDL-C) and apoB-100 and a reduction in aortic lipid deposition and AS lesions in apolipoprotein E-deficient (ApoE-/-) mice fed a high fat diet (HFD). The hepatic levels of Sortilin and apoB-100 expression were also decreased in these treated mice. In conclusion, miR-122 suppresses Sortilin expression and Sortilin-mediated apoB-100 secretion to resist circulating LDL production and aortic AS development, which is enhanced by 6-MC-upregulated miR-122 in the liver.

Development of Sustainable Insecticide Candidates for Protecting Pollinators: Insight into the Bioactivities, Selective Mechanism of Action and QSAR of Natural Coumarin Derivatives against Aphids.

Plants employ abundant toxic secondary metabolites to withstand insect attack, while pollinators can tolerate some natural defensive compounds. Coumarins, as promising green alternatives to chemical insecticides, possess wide application prospects in the crop protection field. Herein, the bioactivities of 30 natural coumarin derivatives against Aphis gossypii were assessed and revealed that 6-methylcoumarin exhibited potent aphicidal activity against aphids but displayed no toxicity to honeybees. Additionally, using biochemical, bioinformatic, and molecular assays, we confirmed that the action mode of 6-methylcoumarin against aphids was by inhibiting acetylcholinesterase (AChE). Meanwhile, functional assays revealed that the difference in action site, which located in Lys585 in aphid AChE (equivalent to Val548 in honeybee AChE), was the principal reason for 6-methylcoumarin being toxic to aphids but safe to pollinators. This action site was further validated by mutagenesis data, which uncovered how 6-methylcoumarin was unique selective to the aphid over honeybee or mammalian AChE. Furthermore, a 2D-QSAR model was established, revealing that the central structural feature was H3m, which offers guidance for the future design of more potent coumarin compounds. This work provides a sustainable strategy to take advantage of coumarin analogues for pest management while protecting nontarget pollinators.

Antifungal Activity of 6-Methylcoumarin against Valsa mali and Its Possible Mechanism of Action.

Valsa canker of apple (VCA) caused by Valsa mali severely affected apple production in east Asia. With the increase in drug resistance, there is an urgent need for efficient and environmentally friendly antifungal agents. Coumarins have attracted much attention due to their excellent antimicrobial activity against plant pathogens. In this study, the antifungal activity of several coumarins against phytopathogenic fungi was evaluated, and then the antifungal activity of the screened 6-MCM against V. mali and its underlying mechanism was further investigated. The results of the in vitro antifungal activity assay showed that some coumarins had significant inhibitory effects on V. mali. Notably, 400 mg/L of 6-MCM had the best antifungal activity of 94.6%. Further experiments showed that 6-MCM slowed down the growth of V. mali mycelia and the germination of spores in a concentration-dependent manner, with EC50 of 185.49 and 54.62 mg/L, respectively. In addition, 6-MCM treatment increased mycelial conductivity, extracellular protein leakage, and MDA content, resulting in damage to the cell membrane. Moreover, 6-MCM significantly reduced the cell wall degrading enzymes secreted by V. mali, including EG, PG and PL, thereby limiting its pathogenic capacity. SEM and TEM results showed that 6-MCM treatment had a significant effect on the morphology and ultrastructure of mycelial cells. Inoculation of isolated apple branches found that the application of 6-MCM effectively inhibited the development of VCA and significantly reduced the incidence. All these results suggest that 6-MCM has the potential as a green substitute for VCA control.

In vitro intestinal permeability studies, pharmacokinetics and tissue distribution of 6-methylcoumarin after oral and intraperitoneal administration in Wistar rats

ABSTRACT 6-Methylcoumarin (6MC) is a semisynthetic coumarin with important in vitro and in vivo anti-inflammatory activity. In order to continue the pre-clinical characterization of this molecule, in vitro intestinal permeability, plasma profile and tissue distribution after oral administration in rats were studied. The permeability of 6MC was evaluated by the Caco-2 cellular model in both the apical-basal (A-B) and basal-apical (B-A) directions. The pharmacokinetics and biodistribution were evaluated in rats after oral and intraperitoneal administration at doses of 200 mg/kg. Transport experiments with Caco-2 cells showed that 6MC presented high permeability at all concentrations evaluated. This finding suggested that 6MC could be transported across the gut wall by passive diffusion. The plasma concentration-time curve showed that the maximum concentration (Cmax) was 17.13 ± 2.90 µg/mL at maximum time (Tmax) of 30 min for the oral route and Cmax 26.18 ± 2.47 µg/mL at 6.0 min for the intraperitoneal administration, with elimination constant of (Ke ) 0.0070 min-1 and a short life half time of (T1/2 ) lower that 120 min. The distribution study showed that 6MC has high accumulation in the liver, and widespread distribution in all the organs evaluated.