Two New Components from an Association of Marine Sponges Poecillastra sp. and Jaspis sp. and Their Inhibitory Effects on Biomarkers for Benign Prostatic Hyperplasia.

Benign prostatic hyperplasia (BPH), characterized by the enlargement of the prostate gland and subsequent lower urinary tract symptoms, poses a significant health concern for aging men with increasing prevalence. Extensive efforts encompassing in vitro and in vivo models are underway to identify novel and effective agents for the management and treatment of BPH. Research endeavors are primarily channeled toward assessing the potential of compounds to inhibit cell proliferation, curb inflammation, and display anti-androgenic activity. Notably, through screening aimed at inhibiting 5-alpha reductase type 2 (5αR2) in human prostatic cells, two acyl compounds (1 and 2) were isolated from a bioactive fraction sourced from an association of marine sponges Poecillastra sp. and Jaspis sp. The complete structure of 1 was determined as (Z)-dec-3-enony (2S, 3S)-capreomycidine, ascertained by JBCA and ECD comparison. While the absolute configurations of 2 remained unassigned, it was identified as a linkage of a 2, 7S*-dihydoxy-9R*-methyloctadecanoyl group with the 2-amino position of a tramiprosate moiety referred to as homotaurine. Evaluation of both compounds encompassed the assessment of their inhibitory effects on key biomarkers (5αR2, AR, PSA, and PCNA) associated with BPH in testosterone propionate (TP)-activated LNCap and RWPE-1 cells.

Anti-Inflammatory and Antiatopic Effects of Rorippa cantoniensis (Lour.) Ohwi in RAW 264.7 and HaCaT Cells.

This study evaluated the effects of Rorippa cantoniensis (Lour.) ohwi extract (RCE) on factors associated with inflammation-related skin lesions in RAW 264.7 and HaCaT cells. RCE inhibited the levels of proinflammatory mediators and cytokines such as nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-6, and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In addition, RCE significantly inhibited the expression of chemokines and cytokines such as MDC/CCL22, TARC/CCL17, RANTES/CCL5, CTSS, IL-6, IL-1ß, and TNF-α in HaCaT cells costimulated by TNF-α and interferon (IFN)-γ in a concentration-dependent manner. These results suggest that RCE attenuated the TNF-α- and IFN-γ-induced release of proinflammatory chemokines and cytokines probably by suppressing the activation of MAPK (JNK and p38), NF-κB, and STAT1 signaling. Moreover, RCE significantly increased the expression of skin components such as hyaluronic acid and aquaporin, which play important roles in the physical and chemical barriers of the skin. These results suggest that RCE has significant anti-inflammatory and antiatopic activities, which may be beneficial for the topical treatment of inflammatory skin disorders.

Correction: Chang et al. Decursinol Angelate Arrest Melanoma Cell Proliferation by Initiating Cell Death and Tumor Shrinkage via Induction of Apoptosis. Int. J. Mol. Sci. 2021, 22, 4096.

The author wishes to make the following correction to this paper [...].

Antibiofilm Activity of Phorbaketals from the Marine Sponge Phorbas sp. against Staphylococcus aureus.

Biofilm formation by Staphylococcus aureus plays a critical role in the persistence of chronic infections due to its tolerance against antimicrobial agents. Here, we investigated the antibiofilm efficacy of six phorbaketals: phorbaketal A (1), phorbaketal A acetate (2), phorbaketal B (3), phorbaketal B acetate (4), phorbaketal C (5), and phorbaketal C acetate (6), isolated from the Korean marine sponge Phorbas sp. Of these six compounds, 3 and 5 were found to be effective inhibitors of biofilm formation by two S. aureus strains, which included a methicillin-resistant S. aureus. In addition, 3 also inhibited the production of staphyloxanthin, which protects microbes from reactive oxygen species generated by neutrophils and macrophages. Transcriptional analyses showed that 3 and 5 inhibited the expression of the biofilm-related hemolysin gene hla and the nuclease gene nuc1.

Decursinol Angelate Arrest Melanoma Cell Proliferation by Initiating Cell Death and Tumor Shrinkage via Induction of Apoptosis.

Melanoma is known to aggressively metastasize and is one of the prominent causes of skin cancer mortality. This study was designed to assess the molecular mechanism of decursinol angelate (DA) against murine melanoma cell line (B16F10 cells). Treatment of DA resulted in growth inhibition and cell cycle arrest at G0/G1 (p < 0.001) phase, evaluated through immunoblotting. Moreover, autophagy-related proteins such as ATG-5 (p < 0.0001), ATG-7 (p < 0.0001), beclin-1 (p < 0.0001) and transition of LC3-I to LC3-II (p < 0.0001) were markedly decreased, indicating autophagosome inhibition. Additionally, DA treatment triggered apoptotic events which were corroborated by the occurrence of distorted nuclei, elevated reactive oxygen species (ROS) levels and reduction in the mitochondrial membrane potential. Subsequently, there was an increase in the expression of pro-apoptotic protein Bax in a dose-dependent manner, with the corresponding downregulation of Bcl-2 expression and cytochrome C expression following 24 h DA treatment in A375.SM and B16F10 cells. We substantiated our results for apoptotic occurrence through flow cytometry in B16F10 cells. Furthermore, we treated B16F10 cells with N-acetyl-L-cysteine (NAC). NAC treatment upregulated ATG-5 (p < 0.0001), beclin-1 (p < 0.0001) and LC3-I to LC3-II (p < 0.0001) conversion, which was inhibited in the DA treatment group. We also noticed a systematic upregulation of important markers for progression of G1 cell phase such as CDK-2 (p < 0.029), CDK-4 (p < 0.036), cyclin D1 (p < 0.0003) and cyclin E (p < 0.020) upon NAC treatment. In addition, we also observed a significant fold reduction (p < 0.05) in ROS fluorescent intensity and the expression of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase-9 (p > 0.010) and cleaved caspase-3 (p < 0.0001). NAC treatment was able to ameliorate DA-induced apoptosis and cell cycle arrest to support our finding. Our in vivo xenograft model also revealed similar findings, such as downregulation of CDK-2 (p < 0.0001) and CDK-4 (p < 0.0142) and upregulation of Bax (p < 0.0001), cytochrome C (p < 0.0001), cleaved caspase 3 (p < 0.0001) and cleaved caspase 9 (p < 0.0001). In summary, our study revealed that DA is an effective treatment against B16F10 melanoma cells and xenograft mice model.

Densazalin, a New Cytotoxic Diazatricyclic Alkaloid from the Marine Sponge Haliclona densaspicula.

Densazalin, a polycyclic alkaloid, was isolated from the marine sponge Haliclona densaspicula collected in Korea. The complete structure of the compound was determined by spectroscopic methods, including 1D and 2D nuclear magnetic resonance techniques, high-resolution mass spectrometry, and comparison of the calculated and measured electronic circular dichroism spectra. Densazalin possesses a unique 5,11-diazatricyclo[7.3.1.02,7]tridecan-2,4,6-triene moiety, which is connected by two linear carbon chains. This compound was derived from the biogenetic precursor bis-1,3-dialkylpyridnium. Densazalin exhibited cytotoxic activity on two human tumor cell lines (AGS and HepG2) in the Cell Counting Kit-8 (CCK-8) bioassay, with IC50 values ranging from 15.5 to 18.4 µM.

Phorbol 12-Myristate 13-Acetate Induced Toxicity Study and the Role of Tangeretin in Abrogating HIF-1α-NF-κB Crosstalk In Vitro and In Vivo.

Phorbol 12-myristate 13-acetate (PMA) is a potent tumor promoter and highly inflammatory in nature. Here, we investigated the toxic effects of PMA on different model system. PMA (10 µg) caused chromosomal aberrations on the Allium cepa root tip and induced mitotic dysfunction. Similarly, PMA caused embryonic and larval deformities and a plummeted survivability rate on zebrafish embryo in a dose-dependent manner. Persistently, PMA treatment on immortalized human keratinocyte human keratinocyte (HaCaT) cells caused massive inflammatory rush at 4 h and a drop in cell survivability at 24 h. Concomitantly, we replicated a cutaneous inflammation similar to human psoriasis induced by PMA. Herein, we used tangeretin (TAN), as an antagonist to counteract the inflammatory response. Results from an in vivo experiment indicated that TAN (10 and 30 mg/kg) significantly inhibited PMA stimulated epidermal hyperplasia and intra-epidermal neutrophilic abscesses. In addition, its treatment effectively neutralized PMA induced elevated reactive oxygen species (ROS) generation on in vitro and in vivo systems, promoting antioxidant response. The association of hypoxia-inducible factor 1-alpha (HIF-1α)-nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) crosstalk triggered by PMA enhanced PKCα-ERK1/2-NF-κB pathway; its activation was also significantly counteracted after TAN treatment. Conclusively, we demonstrated TAN inhibited the nuclear translocation of HIF-1α and NF-κB p65. Collectively, TAN treatment ameliorated PMA incited malignant inflammatory response by remodeling the cutaneous microenvironment.

Protective effect of hypoxylonol C and 4,5,4',5'-tetrahydroxy-1,1'-binaphthyl isolated from Annulohypoxylon annulatum against streptozotocin-induced damage in INS-1 cells.

We evaluated the protective effects of hypoxylonol C and 4,5,4',5'-tetrahydroxy-1,1'-binaphthyl (BNT) isolated from Annulohypoxylon annulatum on pancreatic ß-cell apoptosis, using the ß-cell toxin streptozotocin (STZ). Hypoxylonol C and BNT restored the STZ-induced decrease in INS-1 cell viability in a dose-dependent manner. In addition, treatment of INS-1 cells with 50 µM STZ resulted in an increase in apoptotic cell death, which was observed as annexin V fluorescence intensity. Apoptotic cell death was decreased by co-treatment with 100 µM hypoxylonol C and 100 µM BNT. Similarly, STZ caused a marked increase in the expression of cleaved caspase-8, caspase-3, Bax, and poly (ADP-ribose) polymerase (PARP), as well as a decrease in the expression of B-cell lymphoma 2 (Bcl-2), which was reversed by co-treatment with 100 µM hypoxylonol C and 100 µM BNT. These findings suggest that hypoxylonol C and BNT play an important role in protecting pancreatic ß-cells against apoptotic damage.

Bacillus velezensis: A Valuable Member of Bioactive Molecules within Plant Microbiomes.

Bacillus velezensis is an aerobic, gram-positive, endospore-forming bacterium that promotes plant growth. Numerous strains of this species have been reported to suppress the growth of microbial pathogens, including bacteria, fungi, and nematodes. Based on recent phylogenetic analysis, several Bacillus species have been reclassified as B. velezensis. However, this information has yet to be integrated into a well-organized resource. Genomic analysis has revealed that B. velezensis possesses strain-specific clusters of genes related to the biosynthesis of secondary metabolites, which play significant roles in both pathogen suppression and plant growth promotion. More specifically, B. velezensis exhibits a high genetic capacity for synthesizing cyclic lipopeptides (i.e., surfactin, bacillomycin-D, fengycin, and bacillibactin) and polyketides (i.e., macrolactin, bacillaene, and difficidin). Secondary metabolites produced by B. velezensis can also trigger induced systemic resistance in plants, a process by which plants defend themselves against recurrent attacks by virulent microorganisms. This is the first study to integrate previously published information about the Bacillus species, newly reclassified as B. velezensis, and their beneficial metabolites (i.e., siderophore, bacteriocins, and volatile organic compounds).

Determination of the Absolute Configuration of Polyhydroxy Compound Ostreol B Isolated from the Dinoflagellate Ostreopsis cf. ovata.

Following isolation of the polyhydroxy compound, ostreol B, from cultivated cells of the toxic dinoflagellate Ostreopsis cf. ovata collected in South Korea, 1D and 2D NMR spectroscopy were employed to determine the planar chemical structure of this compound, which contained a tetrahydropyran ring, two terminal double bonds, and 21 hydroxyl groups. The absolute configurations of all stereogenic carbon centers in ostreol B were then determined through a combination of the J-based configuration analysis, rotating frame Overhauser effect correlations, and the modified Mosher method following cleavage of the 1,2-diol bonds. Ostreol B was also found to exhibit moderate cytotoxicity in HepG2, Neuro-2a and HCT-116 cells.

Renoprotective Effects of Hypoxylonol C and F Isolated from Hypoxylon truncatum against Cisplatin-Induced Cytotoxicity in LLC-PK1 Cells.

Although cisplatin is the standard platinum-based anticancer drug used to treat various solid tumors, it can cause damage in normal kidney cells. Protective strategies against cisplatin-induced nephrotoxicity are, therefore, clinically important and urgently required. To address this challenge, we investigated the renoprotective effects of Hypoxylontruncatum, a ball-shaped wood-rotting fungus. Chemical investigation of the active fraction from the methanol extract of H.truncatum resulted in the isolation and identification of the renoprotective compounds, hypoxylonol C and F, which ameliorated cisplatin-induced nephrotoxicity to approximately 80% of the control value at 5 µM. The mechanism of this effect was further investigated using hypoxylonol F, which showed a protective effect at the lowest concentration. Upregulated phosphorylation of p38, extracellular signal-regulated kinases, and c-Jun N-terminal kinases following cisplatin treatment were markedly decreased after pre-treatment with hypoxylonol F. In addition, the protein expression level of cleaved caspase-3 was significantly reduced after co-treatment with hypoxylonol F. These results show that blocking the mitogen-activated protein kinase signaling cascade plays a critical role in mediating the renoprotective effect of hypoxylonol F isolated from H.truncatum fruiting bodies.

Ostreol A: a new cytotoxic compound isolated from the epiphytic dinoflagellate Ostreopsis cf. ovata from the coastal waters of Jeju Island, Korea.

Ostreol A was isolated from cultures of the epiphytic dinoflagellate Ostreopsis cf. ovata from the coastal waters of Jeju Island, Korea. The compound, a non-palytoxin derivative, has a polyhydroxy chain ending with the primary amino group and contains an amide bond, along with two tetrahydropyran rings in the chain. Its chemical structure was elucidated by nuclear magnetic resonance (NMR) spectroscopy methods and confirmed by mass analysis. The compound exhibited significant cytotoxicity in the brine shrimp lethality test at a concentration of 0.9µg/mL.

Characterization and anti-inflammatory effects of iodinated acetylenic acids isolated from the marine sponges Suberites mammilaris and Suberites japonicus.

The previously unknown compounds 1-4, acetylenic acids with one or two iodine atom(s), were isolated from the marine sponges Suberites mammilaris and Suberites japonicus. Their complete structures were determined using NMR and mass spectrometry. The methylated compounds 1a and 2a exhibited a strong NO inhibitory effect on RAW264.7 cells, while methylated 3a and 4a were inactive in RAW264.7 cells, but highly active in BV2 microglia cells.

New constituents from the Korean sponge Plakortis simplex.

Six new cyclic peroxides (1-6) were isolated from the Korean sponge Plakortis simplex, along with two new alkylpyridinium alkaloids (7 and 8). The structures of these compounds were completely determined by a combination of NMR analysis and chemical reactions. Compounds 1-6 exhibited cytotoxic/antifungal activities against RAW264.7 cells and Candida albicans.

Antiamoebic activities of flavonoids against pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba species.

Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established for FLA infections of the CNS, emphasizing the urgent need to discover or develop safe and effective drugs. Flavonoids, natural compounds from plants and plant-derived products, are known to have antiprotozoan activities against several pathogenic protozoa parasites. The anti-FLA activity of flavonoids has also been proposed, while their antiamoebic activity for FLA needs to be emperically determined. We herein evaluated the antiamoebic activities of 18 flavonoids against Naegleria fowleri and Acanthamoeba species which included A. castellanii and A. polyphaga. These flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. Demethoxycurcumin, kaempferol, resveratrol, and silybin (A+B) showed in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species. Apigenin, costunolide, (‒)-epicatechin, (‒)-epigallocatechin, rosmarinic acid, and (‒)-trans-caryophyllene showed selective antiamoebic activity for Acanthamoeba species. Luteolin was more effective for N. fowleri. However, afzelin, berberine, (±)-catechin, chelerythrine, genistein, (+)-pinostrobin, and quercetin did not exhibit antiamoebic activity against the amoeba species. They neither showed selective antiamoebic activity with significant cytotoxicity to C6 glial cells. Our results provide a basis for the anti-FLA activity of flavonoids, which can be applied to develope alternative or supplemental therapeutic agents for FLA infections of the CNS.

Moringa oleifera mitigates ethanol-induced oxidative stress, fatty degeneration and hepatic steatosis by promoting Nrf2 in mice.

BACKGROUND: Moringa oleifera (M. oleifera) is cultivated throughout the world and it is known by numerous regional names and is consumed as medication for various diseases such as hypertension, diabetes, HIV and is potential source of nutrients and natural antioxidants making it among the most useful trees. METHODS: We evaluated the therapeutic potential of M. oleifera on ethanol-induced fatty liver. The mice were treated with 30% ethanol (EtOH) alone or in combination with different concentration of M. oleifera extracts (100, 200 and 400 mg/kg). We performed biochemical estimation for the serum of important liver damage markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and triglyceride (TG). We performed histopathological analysis from the liver tissues of different mice groups. We also performed ELISA assay, western blotting analysis and SPECT imaging to obtain our results. RESULTS: The results for serum (AST, p < 0.0001), (ALT, p < 0.0006) and triglyceride (TG, p < 0.0003) were found to be significantly reduced in all doses of M. oleifera extract treatment groups in comparison with the ethanol group. H&E staining analysis and scoring revealed a significant reduction in lipid droplet accumulation and a significant reduction of liver steatosis (p < 0.0001), lobular inflammation (p < 0.0013), ballooning (p < 0.0004) and immunohistochemistry for TNF-α. M. oleifera also ameliorated ethanol-induced oxidative stress evaluated through MDA (p < 0.0001), H2DCFDA, JC-1 staining and a significant down-regulation of CYP2E1 enzyme (p < 0.0001) in the 200 and 400 mg/kg groups in comparison with EtOH groups. M. oleifera extract also boosted the antioxidant response evaluated through total GSH assay (p < 0.0001) and nuclear translocation of Nrf2. Furthermore, we performed SPECT imaging and evaluated the liver uptake value (LUV) to assess the extent of liver damage. LUV was observed to be lower in the ethanol group, whereas LUV was higher in control and M. olifera treated groups. CONCLUSION: In summary, from this experiment we conclude that M. oleifera extract has the potential to ameliorate ethanol-induced liver damage.

Phragmites australis (Cav.) Trin. ex Steud. Extract Induces Apoptosis-like Programmed Cell Death in Acanthamoeba castellanii Trophozoites.

Acanthamoeba keratitis (AK) is an infectious ocular disease which is difficult to diagnose correctly and cure. Development of an effective and safe therapeutic drug for AK is needed. Our preliminary screening of more than 200 extracts from wild plants collected in Korea suggested the potential amoebicidal activity of Phragmites australis (Cav.) Trin. ex Steud. extract (PAE) against Acanthamoeba species. Here, we aimed to analyze the amoebicidal activity of PAE on Acanthamoeba and its underlying amoebicidal mechanism. PAE induced amoebicidal activity against both A. castellanii and A. polyphaga trophozoites, while it showed low cytotoxicity in human corneal epithelial cells (HCE-2) and human retinal pigment epithelial cells (ARPE-19). Transmission electron microscopy analysis showed subcellular morphological changes, such as increased granules, abnormal mitochondria, and atypical cyst wall formation, in the PAE-treated A. castellanii. Fluorometric apoptosis assay and TUNEL assay revealed apoptosis-like programmed cell death (PCD) in the PAE-treated A. castellanii. The PAE treatment increased reactive oxygen species production and reduced mitochondrial membrane potential in the amoeba. The enhanced expression of autophagy-associated genes was also detected. These results suggested that PAE exerted a promising amoebicidal effect on A. castellanii trophozoites via the PCD pathway. PAE could be a potential candidate for developing a therapeutic drug for AK.

Protective Effects of Oenothera biennis against Hydrogen Peroxide-Induced Oxidative Stress and Cell Death in Skin Keratinocytes.

BACKGROUND: Oenothera biennis (evening primrose) produces bioactive substances with a diverse range of pharmacological functions. However, it is currently unknown whether extract prepared from the aerial parts of O. biennis (APOB) can protect the skin against oxidative stress. OBJECTIVE: The aim of this study is to investigate the protective effects of APOB against oxidative stress-induced damage in human skin keratinocytes (HaCaT) and elucidate the underlying mechanisms. METHODS: We pretreated HaCaT cells with various concentrations of APOB or the antioxidant N-acetyl-L-cysteine before applying H2O2. We then compared the cell viability, intracellular reactive oxygen species (ROS) production, and DNA and mitochondrial damage between pretreated and untreated control cells using a range of assays, flow cytometry, and Western blot analysis and also examined the reducing power and DPPH free radical scavenging activity of APOB. RESULTS: APOB pretreatment significantly increased cell viability, effectively attenuated H2O2-induced comet tail formation, and inhibited H2O2-induced phosphorylation of the histone γH2AX, as well as the number of apoptotic bodies and Annexin V-positive cells. APOB was found to have high reducing power and DPPH radical scavenging activity and also exhibited scavenging activity against intracellular ROS accumulation and restored the loss of mitochondrial membrane potential caused by H2O2. APOB pretreatment almost totally reversed the enhanced cleavage of caspase-3, the degradation of poly (ADP-ribose)-polymerase (PARP), DNA fragmentation that usually occurs in the presence of H2O2, and increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme that is associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). CONCLUSIONS: APOB can protect HaCaT cells from H2O2-induced DNA damage and cell death by blocking cellular damage related to oxidative stress via a mechanism that affects ROS elimination and by activating the Nrf2/HO-1 signaling pathway.

Distribution and diversity of bacterial endophytes from four Pinus species and their efficacy as biocontrol agents for devastating pine wood nematodes.

In this study, we isolated a total of 238 culturable putative bacterial endophytes from four Pinus species (Pinus densiflora, P. koraiensis, P. rigida, and P. thunbergii) across 18 sampling sites in Korea. The samples were cultured in de Man Rogosa Sharpe and humic acid-vitamin agar media. These selective media were used to isolate lactic acid bacteria and Actinobacteria, respectively. Analysis using 16S ribosomal DNA sequencing grouped the isolated putative bacterial endophytes into 107 operational taxonomic units (OTUs) belonging to 48 genera. Gamma-proteobacteria were the most abundant bacteria in each sampling site and three tissues (needle, stem and root). The highest OTU richness and diversity indices were observed in the roots, followed by stem and needle tissues. Total metabolites extracted from three isolates (two isolates of Escherichia coli and Serratia marcescens) showed significant nematicidal activity against the pine wood nematode (Bursaphelenchus xylophilus). Our findings demonstrated the potential use of bacterial endophytes from pine trees as alternative biocontrol agents against pine wood nematodes.

Decursinol angelate ameliorates 12-O-tetradecanoyl phorbol-13-acetate (TPA) -induced NF-κB activation on mice ears by inhibiting exaggerated inflammatory cell infiltration, oxidative stress and pro-inflammatory cytokine production.

Decursinol angelate (DA) is a pyranocoumarin purified from the roots of Angelica gigas. Here, we synthesized DA and determined its anti-inflammatory potential on TPA-induced mice ear inflammation. First, we evaluated the non-toxic behaviour of DA on HaCaT cells. Additionally, we observed the free radical scavenging potential of DA at 60 µM to be 50%. This finding was further supported by nitric oxide assay, malondialdehyde assay, H2DCFDA staining and western blotting analysis of antioxidant enzymes. DA also suppressed the activation and polarization of macrophage phagocytic activity on RAW 264.7 cells. We further evaluated the expression of ICAM-1, MCP-1, MIP-2 and MIP-1ß on in-vivo model system. Consequently, DA significantly reduced the production of NF-κB and COX-2 induced proinflammatory cytokine levels on TPA induced ear edema. Inhibition of MAPK and transcriptional factor NF-κB was also validated by western blotting analysis of p-ERK, p-p38, IKKα, IKKγ, IκBα, NF-κB-p65. Immunohistochemistry and immunofluorescence staining of NFκB-p65, TNF-α and IL-1ß were also performed to support the findings. Conclusively, these results suggest that topical administration of DA significantly inhibited the expression of pro-inflammatory cytokines by blocking the canonical NF-κB and MAPK pathway. Therefore, we suggest DA as a potent therapeutic compound against skin inflammation related diseases.