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Isoquercitrin (ISO), one of phytochemical isolated from aerial parts of Aster yomena, has been reported to have antifungal activity. However, the synergistic effect and the mechanism of ISO in combination with conventional antifungal agents are poorly understood. Therefore, synergistic antifungal effect between ISO and conventional antifungal agents was investigated. ISO at non-antifungal concentration interacts synergistically with amphotericin B (AMB) and fluconazole (FLC), but the combination with flucytosine (5-FC) showed no interaction. ISO disrupted an antioxidant system by inhibiting the activity of superoxide dismutase. This redox imbalance was shown to induce intracellular reactive oxygen species (ROS) accumulation and oxidative stress. ISO combined with FLC caused metacaspase activation and DNA condensation, markers of apoptosis, higher than the combination with ISO/AMB. In contrast, ISO with AMB synergistically stimulated membrane permeabilization compared to ISO/FLC. Scavenging ROS consequently reduced the synergy-induced apoptosis and membrane permeabilization, indicating combinations induced ROS were associated with the synergy effect of ISO. In conclusion, AMB and FLC enhanced the antifungal potency of ISO through oxidative stress when used in synergy. © 2018 IUBMB Life, 71(1):283-292, 2019., (© 2018 International Union of Biochemistry and Molecular Biology.)

This study analyzes the antifungal properties of (-)-nortrachelogenin and elucidates its mode of action against pathogenic fungi. We performed susceptibility tests against several pathogenic fungi and verified the absence of hemolysis against human erythrocytes. Its antifungal activity increased reactive oxygen species (ROS) in response to intracellular stress and increased concentrations of both intracellular and extracellular trehalose without causing hemolysis. In addition, a cell wall regeneration study indicated its action on the cytoplasmic membrane. A cell surface study using 3,3(')-dipropylthiacarbocyanine iodide [DiSC3(5)] and 1,6-diphenyl-1,3,5-hexatriene (DPH) demonstrated dissipation of the cytoplasmic membrane at high concentrations. Our study revealed a disturbance in the membrane at higher concentrations and externalization of phosphatidylserine in a dose-dependent manner, affecting other intracellular responses. Furthermore, we investigated the late stage of apoptosis using TUNEL and 4('),6-diamidino-2-phenylindole (DAPI) assays. (-)-Nortrachelogenin-treated cells underwent apoptosis which was triggered by mitochondrial dysfunction via depolarization of the mitochondrial membrane, release of cytochrome c and calcium ion signaling, resulting in the activation of metacaspases. Different concentrations of (-)-nortrachelogenin induced membrane disruption and caspase-dependent apoptosis., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The phytochemical (+)-Medioresinol, a furofuran type lignan identification and isolation on the stem bark of Sambucus williamsii, which is a folk medicinal plant used in traditional medicine. (+)-Medioresinol is known to possess a lesishmanicidal activity and cardiovascular disease risk reduction but its antifungal effects have not yet been identified. In this study, to confirm (+)-Medioresinol's antifungal properties and mode of action, we observed morphological and physiological change in Candida albicans. In cells exposed to (+)-Medioresinol, arrested the cell cycle and intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased. The increase of ROS induced oxidative stress and the mitochondria dysfunction which causes release of pro-apoptotic factors. We investigated a series of characteristic cellular changes of apoptosis by using various apoptosis detection methods. We report here for the first time that (+)-Medioresinol has effects on mitochondria and induced the accumulation of ROS in C. albicans cells. We demonstrated that one of the important features of apoptosis, mitochondrial membrane depolarization is caused by ROS. Substantially, we investigated the release of cytochrome c, which is one of the factors of metacaspase activity. We also show that the effects of (+)-Medioresinol are mediated at an early stage in apoptosis acting on the plasma membrane phosphatidylserine externalization. In addition, (+)-Medioresinol induced apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC). In late stage of confirmation of diagnostic markers in yeast apoptosis include the effects of nucleus morphological change, DNA fragmentation and condensation by influence of oxidative stress. These apoptotic phenomena represent that oxidative stress and mitochondria dysfunctions by inducing the phytochemical (+)-Medioresinol must be an important factors of the apoptotic process in C. albicans. These results support the elucidation of the underlying antifungal mechanisms of (+)-Medioresinol., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Amentoflavone was isolated from an ethyl acetate extract of the whole plant of Selaginella tamariscina. It is a traditional herb for the therapy of chronic trachitis and exhibits some anti-tumor activity. Previously, we confirmed the antifungal effects of amentoflavone. The objective of this study was to investigate the antifungal mechanism(s) of amentoflavone, such as mitochondria-mediated apoptotic cell death. The cells that were treated with amentoflavone exhibited a series of cellular changes that were consistent with apoptosis: externalization of phosphatidylserine, DNA and nuclear fragmentation, accumulation of intracellular reactive oxygen species (ROS) and hydroxyl radicals, and activation of metacaspase. In addition, diagnostic markers of apoptosis, including the reduction of mitochondrial inner-membrane potential and the release of cytochrome c from mitochondria, were observed. These phenomena are important changes in mitochondria-mediated apoptosis. Furthermore, the effect of thiourea as hydroxyl radical scavenger on amentoflavone-induced apoptosis was evaluated. A hydroxyl radical is a more active ROS species. Mitochondrial dysfunction was inhibited, which was indicated by decreased levels of intracellular hydroxyl radicals. Taken together, our results present the first evidence that amentoflavone induces apoptosis in C. albicans cells and is associated with the mitochondrial dysfunction. Besides, amentoflavone-induced hydroxyl radicals may play a significant role in mitochondria-mediated apoptosis.

Amentoflavone, a biflavonoid from Selaginella tamariscina, is known to possess several bioactivities such as antitumor, anti-inflammatory, and antifungal effects. However, the mechanism of the anticancer effects of amentoflavone on human cervical cancer cells has not been studied in detail. In this study, we demonstrated that amentoflavone induces apoptosis in SiHa and CaSki cervical cancer cells by suppressing human papillomavirus protein E7 expression. The cyclins and tumor suppressors were modulated by amentoflavone in SiHa and CaSki human cervical cancer cells: cyclin and hyperphosphorylated retinoblastoma (p-pRb) were down-regulated, whereas cyclin-dependent kinase inhibitors and p53 were enhanced. Amentoflavone up-regulated peroxisome proliferator-activated receptor γ (PPARγ) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression levels while inhibiting E7-mediated cyclooxygenase-2 (COX-2)/interleukin-32 (IL-32) expressions were downregulated, and Akt phosphorlylation was decreased in an amentoflavone-induced apoptotic process, suggesting that amentoflavone may be a PPARγ activator. Additionally, the expression of the anti-apoptotic factor Bcl-2 was decreased, whereas that of the well-known apoptotic factor Bax was increased, thereby releasing cytochrome c into cytosol in amentoflavone-treated cervical cancer cells. Furthermore, amentoflavone treatment led to the activation of caspase-3 and -9 and proteolytic cleavage of poly(ADP-ribose) polymerase. The expression level of the extrinsic death receptor Fas (CD95) was not altered by amentoflavone treatment. When these findings are taken together, the biflavonoid amentoflavone activates PPARγ/PTEN expressions and induces apoptosis via suppressing E7 expression, cell cycle arrest at sub-G₁ phase, and mitochondria-emanated intrinsic pathways in SiHa and CaSki human cervical cancer cells. These findings suggest that amentoflavone has potential for development as a therapeutic agent for human cervical cancer.

The antifungal effects and mechanisms of Styraxjaponoside C were investigated. Styraxjaponoside C was active against several human pathogens, including Candida albicans. Styraxjaponoside C induced a series of cellular changes characteristic of apoptosis in C. albicans, including increased reactive oxygen species (ROS) production, measured by DHR-123 staining; phosphatidylserine externalization, visualized by Annexin V staining; DNA fragmentation, as seen by TUNEL; and plasma membrane depolarization, observed by DiBAC(4)(3) staining. The plasma membrane depolarization is likely to be associated with production of ROS. The current study suggests that Styraxjaponoside C exerts an antifungal effect by promoting apoptosis.

Plantainoside D (PD), was isolated from the leaves of Picrorhiza scrophulariiflora (Scrophulariaceae). The anti-oxidative activity of PD was evaluated based on scavenging effects on hydroxyl radicals and superoxide anion radicals. Adriamycin (ADR) is a potent anti-tumor drug known to cause severe cardiotoxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity has not been understood. This study was undertaken to investigate the protective effect of PD against ADR-induced apoptosis. In vitro, ADR caused dose-dependent toxicity in H9c2 cardiac muscle cells. Pre-treatment of the cardiac muscle cells with PD significantly reduced ADR-induced apoptosis of cardiac muscle cells. PD inhibited the ROS produced by ADR in the cardiac muscle cells. As well, PD increased GSH(glutathione), compared with ADR. In response to ADR, NF-kappaB was activated in H9c2 cells. However the treatment of PD reduced the activation of NF-kappaB. We also observed that the NF-kappaB inhibitor, PDTC, inhibited the cytotoxic effect on ADR-induced apoptosis in cardiac muscle cells. In parallel, IkappaBalpha-dominant negative plasmid-overexpression abrogated ADR-induced apoptosis in H9c2 cardiac muscle cells. In conclusion, these results suggest that Plantaionoside D can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS generation and NF-kappaB activation. The pure compound PD can be a potential candidate agent which protects cardiotoxicity in ADR-exposed patients.

Rosmarinic acid (RA) is a naturally occurring polyphenolic and is found in several herbs in the Lamiaceae family, such as, Perilla frutescens. ADR is a potent anti-tumor drug, but is unfortunately potently cardiotoxic. This study was undertaken to investigate the inhibitory effect of RA on ADR-induced apoptosis in H9c2 cardiac muscle cells at a mechanistic level. In vitro, ADR significantly decreased the viabilities of H9c2 cells, and this was accompanied by apoptotic features, such as a change in nuclear morphology and caspase protease activation. RA was found to markedly inhibit these apoptotic characteristics by reducing intracellular ROS generation and by recovering the mitochondria membrane potential (delta psi). In addition, RA reversed the downregulations of GSH, SOD and Bcl-2 by ADR. In the present study, ADR was found to activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), transcriptional factor-activator-protein (AP)-1. We found that c-fos, Jun-B, Jun-D and p-c-Jun were super shifted by ADR, indicating that these proteins have an important role in the ADR-induced AP-1 activation. The inhibitions of JNK and ERK using appropriate inhibitors or dominant negative cell lines reduced ADR-induced apoptosis in H9c2 cardiac muscle cells. Taken together, these results suggest that RA can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells by inhibiting ROS generation and JNK and ERK activation. Thus, we propose that RA should be viewed as a potential chemotherapeutic that inhibits cardiotoxicity in ADR-exposed patients.

Abstract Ethnopharmacological relevance Aster yomena , a perennial herb that grows mainly in South Korea, has been employed in the traditional temple food for antibiotic efficacy. Recently, it was reported that apigenin isolated from A. yomena has a physical antifungal mechanism targeting membrane against Candida albicans. Aim of the study Our study aimed to investigate the biochemical responses underlying the antifungal activity of apigenin isolated from A. yomena due to lack studies reporting the investigation of intracellular responses of apigenin in C. albicans. Materials and methods Apigenin was isolated from the aerial parts of A. yomena. To evaluate apigenin-induced inhibitory effects and membrane damages, the measurement of the cell viability assay and the flux of cytosolic components were performed with at various concentrations. Intracellular external potassium and calcium levels were assayed by an ion-selective electrode meter, Fura2-AM and Rhod2-AM, respectively. Mitochondrial dysfunctions were analyzed by using JC-1, Mitotracker Green FM, and MitoSOX Red dye. H 2 DCFDA, glutathione, and MDA assay were used to detect oxidative damage. Also, flow cytometry was carried out to detect apoptotic hallmarks using Annexin V-PI, TUNEL, and FITC-VAD-FMK staining. Tetraethylammoniumchloride (TEA), Ruthenium red (RR), and N -acetylcysteine (NAC) were used as a potassium channel blocker, mitochondrial calcium uptake inhibitor, and reactive oxygen species (ROS) scavenger, respectively. Results We confirmed that there was no decrease of cell survival percentages in crude extracts of A. yomena treatment, however, only isolated apigenin has the antifungal effect in C. albicans. Apigenin triggered a dose-dependent mitochondrial calcium uptake followed by mitochondrial dysfunction, loss of the membrane potential and an increase in the mitochondrial mass and ROS. Apigenin also induced intracellular redox imbalance as indicated by the ROS accumulation, glutathione oxidation, and lipid peroxidation. Interestingly, NAC failed the restore the mitochondrial calcium levels and thus alleviate the mitochondrial damages, however, RR reduced the apigenin-induced redox imbalance. Furthermore, apigenin induced apoptosis activation marked by the phosphatidylserine exposure, DNA fragmentation, and caspase activation. The pro-apoptotic effect of apigenin was counteracted by RR and NAC pretreatment. In particular, RR significantly reduced the pro-apoptotic responses. Conclusions Apigenin isolated from A. yomena induced mitochondrial-mediated apoptotic pathway, and mitochondrial calcium signaling is main factor in its pathway in C. albicans. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Minimum inhibitory concentration (MIC) is defined as the lowest concentration of a compound that completely inhibits microbial growth. Antibacterial mechanisms of compounds have been investigated at their sub-MICs as well as at their MIC. In this study, the effects of sub-MIC and MIC of isoquercitrin on Escherichia coli were investigated. The antibacterial effect of isoquercitrin was tested using the microdilution method. Sub-MICs of isoquercitrin induced the production of reactive oxygen species and depletion of glutathione. The oxidative effects induced by sub-MICs of isoquercitrin could be prolonged, finally resulting in apoptosis-like death. DNA fragmentation and phosphatidylserine externalization, which are regarded as the hallmarks of apoptosis, were evaluated using the TUNEL assay and Annexin V staining, respectively. Furthermore, isoquercitrin induced the peroxidation of membrane lipids and inner membrane permeabilization at both its sub-MIC and MIC. This suggested membrane damage in response to lipid oxidation. The uptake of membrane impermeable dyes, propidium iodide and calcein, demonstrated that isoquercitrin damaged the cell membrane at concentrations higher than its MIC. Thus, isoquercitrin induced apoptosis-like death and dysregulation of cell membrane dynamics. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a gram-negative bacterium that is frequently related to natural resistance to many drugs. In this work, the inhibition of growth against P. aeruginosa and multidrug-resistant P. aeruginosa (MDRPA) isolated from patients at Kyungpook National University was confirmed for hibicuslide C, essential oil components from Abutilon theophrasti. Hibicuslide C has antifungal activity with membrane disruption and apoptotic response against Candida albicans. However, its antibacterial activity was not reported yet. Cells treated with hibicuslide C was showed that its antipseudomonal activity is related to gDNA fragmentation and damage by TUNEL and gDNA electrophoresis. Furthermore, hibicuslide C worked synergistically with fluoroquinolones and rifampicin against MDRPA regardless of the ATP-associated mechanism. The antibiofilm activity possessed sole-resulting tissue culture plate method; besides that, the antibiofilm activity of other antibiotics was supported in particular MDRPA. The essential oil components like hibicuslide C may have antipseudomonal activity and, furthermore, increase in bacterial antibiotic susceptibility. [ABSTRACT FROM AUTHOR]

Isoquercitrin has received much interest due to its various biological activities and exerts the antifungal effect. Therefore, we aimed to discover the novel antifungal mechanism of isoquercitrin in Candida albicans , showing apoptotic cell death. Using 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining, the levels of reactive oxygen species (ROS) were measured and isoquercitrin revealed different effects in a concentration-dependent manner: ROS scavenging effect (low concentration) and ROS forming effect (high concentration). Furthermore, we used the antioxidant, N-acetylcysteine (NAC) to discriminate the influence of ROS in DNA fragmentation and caspase activation. The green fluorescence of TUNEL and FITC-VADFMK was detected in high concentration-treated cells, whereas there was no fluorescence in both low concentration-treated cells and NAC-treated cells. These results demonstrated that ROS generation is the crucial factor in isoquercitrin-induced apoptosis. Thus, isoquercitrin induces ROS-mediated apoptosis in C . albicans. [ABSTRACT FROM AUTHOR]

Abstract: Adriamycin is a potent antitumor drug that is known to cause severe cardiotoxicity. This study examined the protective effect of calceolarioside on adriamycin-induced cardiomyocyte toxicity. Calceolarioside significantly inhibited the adriamycin induced cell death and caspase-3 activation, which may be explained by the increase in Bcl-2 expression and the inhibition of Bax expression. Calceolarioside increased the expression of the antioxidant molecules and decreased the level of intracellular reactive oxygen species. Catalase, glutathione, N-acetylcysteine, Mannitol and Mn-TBAP (manganese (III) tetrakis-(4-benzoic acid) porphyrin) significantly inhibited the H9c2 cell death induced by adriamycin. Calceolarioside significantly inhibited H9c2 cell death, and was more effective than that observed with the other antioxidants, including probucol, ascorbic acid, and alpha-tocopherol. Overall, these results suggest that calceolarioside can inhibit adriamycin-induced apoptosis in H9c2 cardiomyocyte by inhibiting the generation of reactive oxygen species. Calceolarioside may be a potential candidate agent that inhibits cardiomyocyte-toxicity in adriamycin-exposed patients. [Copyright &y& Elsevier]

Abstract: This study examined the effect of Saeng-Ji-Hwang (SJH: Radix Rehmanniae) on cardiac muscle cells. Adriamycin-exposed H9C2 cardiac muscle cells were treated with a water extract of SJH. The adriamycin induced cell death and caspase-3 activation were significantly inhibited by SJH (2 mg/ml), which can be explained by the increase in Bcl-2 expression and the inhibition of Bax expression. Adriamycin reduced the Mn-SOD protein expression level in H9C2 cardiac muscle cells but a SJH treatment partially but significantly reversed this effect. Manganese (Mn)-TBAP or Mn-TMyM— mitochondria-specific SOD mimetic agent— reduced the adriamycin-induced cytotoxicity. It was also shown that SJH inhibits the release of H2O2 and prevents lipid peroxidation in the presence of adriamycin. This study examined the intracellular GSH level, which showed that adriamycin significantly decreased the intracellular GSH level but SJH increased it. BSO, a selective inhibitor of glutamyl cysteinyl ligase, which is a rate-limiting enzyme in GSH synthesis, did not affect the viability of the cardiac muscle cells. However, a combination of BSO with SJH in the presence of adriamycin reversed the SJH-induced protection. Overall, the results suggest that SJH-associated Mn-SOD and GSH are important factors in the mechanism of the SJH-induced protective mechanism in H9C2 cardiac muscle cells. [Copyright &y& Elsevier]